Write Answers for the questions from the assigned Chapters

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The Puerperium & Lactation

Parturition

Fetal Attachment & Gestation

Early Embryogenesis & Maternal Recognition of Pregnancy

Ovulation & Fertilization

Cyclicity

Regulation of Reproduction

Tract Function

Puberty

Prenatal Development

Spermatogenesis

Regulation of Reproduction

Tract Function

Puberty

Prenatal Development

Take Home Message In the adult male, GnRH, LH and testosterone are secreted in pulses that occur eve1y

several hours. Follicle stimulating hormone is released in smaller pulses of longer duration. Spermatozoa are produced by the testes by a process called spermatogenesis that requires 5 to 9 weeks, depending on the species. The number of sperm produced each day is indepen- dent of the number ejaculated. Spermatogenesis is a process involving sequential mitotic and meiotic divisions and concludes after differentiation of spherical spermatids into highly specialized spermatozoa. Spermatozoa are released continually from the seminiferous epi- thelium in post-pubertal males.

Endocrine Control/Regulation is Different than in the Female

Before spemmtozoa can be produced, certain endocrine requirements must be met. They are: I) adequate secretion of GnRH from the hypothalamus; 2) FSH and LH secretion from the anterior lobe of the pituitary and 3) secretion of gonadal steroids (testos- terone and estradiol). Recall from Chapter 6 that the hypothalamus in the male does not develop a surge center. The discharge ofGnRH from the hypothalamus in the male occurs in frequent, intermittent episodes that occur throughout the day and night. These short-lived bursts of GnRH last for only a few minutes and cause discharges of LH that fo llow almost immediately after the GnRH episode. The episodes of LH last from l 0 to 20 minutes and occur between 4 to 8 times every 24 hours. Concentrations ofFSH are lower, but the pulses are oflonger duration than LH because of the relatively constant secretion of inhibin by the adult testis and the longer half-life of FSH (See Figure 10-1 ).

Luteinizing hormone acts on the Leydig cells within the testes. These cells, named after the Gem1an anatomist Franz von Leydig, are analogous to the cells of the theca intema of antral follicles in the ovmy . They contain membrane-bound receptors for LH. When LH binds to their receptors, Leydig cells synthesize pro- gesterone, most of which is converted to testosterone. Blood LH is elevated for about 30 to 75 minutes. The Leydig cells synthesize and secrete testosterone less than 30 minutes after the onset of an LH episode. The

response (testosterone secretion) by Leydig cells is short and secretion is pulsatile, lasting for a period of 20 to about 60 minutes (See Figure I 0-2).

Successful testis function requires: • pulsatile GnRH secretion (every 3-6/trs) • high concentrations of testosterone in the seminiferous tubule

•low concentrations oftestosterone in a systemic blood

• adequate LH receptors in Leydig cells

Pulsatile discharge of LH is important for nomml testicular function. The pulsati le nature ofLH secretion prevents sustained concentrations of LH to which the Leydig cells become refractory (unrespon- sive or not yielding to treatment). A refractory condi- tion is thought to be caused by reduction in the number of LH receptors in the Leydig cells. As a result oflow receptor numbers, reduced secretion of testosterone by Leydig cells follows. Pulsatile LH secretions optimize LH receptor numbers and testosterone secetion by Leydig cells.

Normally, intratesticular concentrations of testosterone are 100-500 times higher than that of systemic blood. High concentrations of testosterone are required for normal spermatogenesis. When tes- tosterone from the testis mixes with peripheral blood,

Production of normal numbers of fertile spermatozoa requires: • endocrine regulation of the testis • mitotic divisions of spermatogonia • meiotic divisions resulting in haploid spermatids • morphologic transformation of spermatids into spermatozoa

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The Puerperium & Lactation

Parturition

Fetal Attachment & Gestation

Early Embryogenesis & Maternal Recognition of Pregnancy

Ovulation & Fertilization

Cyclicity

Regulation of Reproduction

Tract Function

Puberty

Prenatal Development

Spermatogenesis

Regulation of Reproduction

Tract Function

Puberty

Prenatal Development

Take Home Message In the adult male, GnRH, LH and testosterone are secreted in pulses that occur eve1y

Endocrine Control/Regulation is Different than in the Female

response (testosterone secretion) by Leydig cells is short and secretion is pulsatile, lasting for a period of 20 to about 60 minutes (See Figure I 0-2).

Successful testis function requires: • pulsatile GnRH secretion (every 3-6/trs) • high concentrations of testosterone in the seminiferous tubule

•low concentrations oftestosterone in a systemic blood

• adequate LH receptors in Leydig cells

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204 Spermatogenesis

Figure 10-1. Relationship Between GnRH, LH and FSH in the Male Cl) 1: 0 E 111 LH :1.. 1: oo .s:::·-...

-ot..s 0 :1.. ot: -CI) .Ou Cl) 1: > 0 ·.p u t..s Cl)

LH LH

/\ G n RH

LH GnRH causes the release of LH and FSH. Episodes of all three hormones occur between 4 and 8 times in 24 hours. The lower FSH profile, when compared to LH , is due to inhibin secretion by Sertoli cells. Also, the greater duration of the FSH episode is probably due to its longer half-life (1 00 min) when compared to LH

0 3 6 9 12 IS (30 min).

Time (hours)

it is diluted over 500 times. This is important because it keeps systemic concentrations well below that which would cause down-regulation of the GnRH/LH feed- back system. For example, if LH pulses were long (hours), Leydig cells would secrete testosterone for hours rather than minutes. This would likely result in a metabolic overload for testosterone clearance and tes- tosterone would exert a sustained negative feedback on the GnRH neurons in the hypothalamus. The net effect would be significantly reduced LH secretion, followed by severely reduced testosterone secretion.

The role of the pulsatile nature of testosterone is not f·ully understood. It is thought that a chronically high systemic concentration of testosterone removes the negative feedback on FSH. Sertoli cell function is FSH dependent. Thus, their function is compromised when FSH is reduced. The periodic reduction in tes- tosterone removes the negative feedback on FSI-1 (See Figure 10-3 ).

In addition to secretion of testosterone by the Leydig cells, the testes also secrete estradiol and other estrogens. The stallion and the boar secrete large amounts of estrogens (both free and in conjugated form). In fact, urinary estrogens in the male are signifi- cantly higher than urinary estrogens in pregnant mares and sows. These high concentrations of estradiol seem to be of little consequence, s ince they are secreted as molecules with low physiologic activity.

Ley dig cells are the male equivalent ofthefol/icular theca interna cells.

Sertoli cells are the male equivalent of the follicular granulosa! cells.

Figure 10-2. Typical Peripheral Concentrations of Blood LH and Testosterone (T) in the Male

-E 4 -DO r::: J: 2 .....

3 6 9

Time (hours)

Q) LH is elevated for a period r::: of 0.5 to 1.25 hours, while 10 o-s..- the subsequent testoster-Q) E ._._ one (T) episode lasts for 1111:).0

0 c: 0.5 to 1.5 hours . ... _ 5 Ill Q)

Spermatogenesis 205

Figure 10-3. Interrelationships Among Hormones Produced by Sertoli Cells, Leydig Cells, the Hypothalamus and the Anterior Lobe of Pituitary

soox dilution

by systemic circulat ion

\ LH

The Sertoli cells secrete inh ib in that exerts a negative feedback on the anterior lobe of the pituitary to directly sup- press FSH secretion.

( Blue spheres = spe r matogonia ; Re d sphe res = primary spermatocytes; Brown spheres = secondary spermatocytes; Black spheres = spermatids Testosterone (T) secreted by the Leydig cells is trans- LH binds to receptors in the interstitial ported into the Sertoli cells where it is converted to cells of Leydig and FSH binds to Sertoli dihydrotestosterone (DHT) and also estradiol (E2 ). cells. Leydig cells secrete testosterone Testosterone and E2 are transported by the blood to that is transported to the adjacent vas- the hypothalamus where they exert a negative feed- culature and the Sertoli cells where Tis back on the GnRH neurons. converted to DHT.

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204 Spermatogenesis

Figure 10-1. Relationship Between GnRH, LH and FSH in the Male Cl) 1: 0 E 111 LH :1.. 1: oo .s:::·-...

-ot..s 0 :1.. ot: -CI) .Ou Cl) 1: > 0 ·.p u t..s Cl)

LH LH

/\ G n RH

0 3 6 9 12 IS (30 min).

Time (hours)

Ley dig cells are the male equivalent ofthefol/icular theca interna cells.

Sertoli cells are the male equivalent of the follicular granulosa! cells.

Figure 10-2. Typical Peripheral Concentrations of Blood LH and Testosterone (T) in the Male

-E 4 -DO r::: J: 2 .....

3 6 9

Time (hours)

Q) LH is elevated for a period r::: of 0.5 to 1.25 hours, while 10 o-s..- the subsequent testoster-Q) E ._._ one (T) episode lasts for 1111:).0

0 c: 0.5 to 1.5 hours . ... _ 5 Ill Q)

Spermatogenesis 205

Figure 10-3. Interrelationships Among Hormones Produced by Sertoli Cells, Leydig Cells, the Hypothalamus and the Anterior Lobe of Pituitary

soox dilution

by systemic circulat ion

\ LH

The Sertoli cells secrete inh ib in that exerts a negative feedback on the anterior lobe of the pituitary to directly sup- press FSH secretion.

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206 Spermatogenesis

Sertoli cells convert testosterone to estradiol utilizing a mechanism identical to the granulosa) cells ofthe antral follicle in the female. The exact role of estradiol in male reproduction is poorly understood, but there is little doubt that this hormone has a negative feedback role on the hypothalamus. Testosterone and estradiol in the blood act on the hypothalamus and exert a negative feedback on the secretion of GnRH and, in tum, LH and FSH are reduced. Therefore, high concentrations of estradiol result in suppression of GnRH and LH discharges (See Figure 1 0-3). In addition to terone to estradiol, Sertoli cells also secrete mh1bm that, as in the female, suppresses FSH secretion from the anterior lobe of the pituitary. The importance of inhibin and suppressed FSH release is not clear in the male.

The goals of spermatogenesis are to: • provide a continual supply of male gametes (up to decades) through stem cell renewal

• provide genetic diversity

• provide billions of sperm each day (domestic animals) to maxi- mize reproduction by both natural service and artificial insemination

• provide an immunologically privileged site where developing germ cells are not destroyed by the immune system

Figure 10-4. Scanning Electron Micrograph of Testicular Parenchyma in the Stallion

(Courtesy of Dr. Larry Texas The A:nerican Society for Reproductive Med1cme. Fertil. and Stenl., 1978. 29.208-215)

Seminiferous tubules (ST) containing developing germ cells (GC) are surrounded by a basement Flagella (F) from developing spermatids can be observed protruding into of some tubules. The rntersttt1al compartment contains Leydig cells (LC), blood vessels (BV) and connective t1ssue (CT).

Spermatogenesis 207

Spermatogenesis =proliferation + meiosis + differentiation

Spermatogenesis is the Process of Producing Spermatozoa

Spermatogenesis takes place entirely within the seminiferous tubules (See Figure 1 0-4) and consists of all cell divisions and morphologic changes that oc- cur to developing gem1 cells. (See Figures I 0-5 and 3-16).

The process of spermatogenesis can be subdi- vided into three phases. The first phase, designated the proliferation phase, consists of all mitotic divisions of spermatogonia. Several generations of A-spermatogo- nia undergo mitotic divisions, generating a large number of B-spermatogonia (See Figure I 0-5). An important part of the proliferation phase is stem cell renewal. Loss of intercellular bridges allows some spennatogo-

nia to revert to stem cells (spetmatogonial stem cells) providing continual renewal of these stem cells from which new spermatogonia can develop.

The meiotic phase begins with primary spetmatocytes. During meiosis I, genetic diversity is guaranteed by DNA replication and crossing over dur- ing the production of secondary spermatocytes. From a genetic perspective no two sperm are identical. Conclu- sion of the meiotic phase (the second meiotic division) produces haploid (lN) spermatids.

The third or final phase of spermatogenesis is the differentiation phase. No further cell divisions take place during this phase. The differentiation phase has commonly been referred to as "spermiogenesis" in reproductive physiology li terature. During the differ- entiation phase, a spherical undifferentiated spermatid

Figure 10-5. Typical Sequence of Spermatogenesis in Mammals Spermatogonia (A1-At, I and B) undergo a series of mitotic divisions (Mit) and the last mitotic division gives rise to primary spermatocytes that enter meiosis. This series of mitotic divisions allows for continual prolif- eration of spermatogonia and replacement of A1 spermatogonia.

I Proliferation I

Basement membrane

Spermatogonia (A2) D Spermatogonia (A3) IJ Number of divisions depends on

species

Spermatogonia ( I ) IJI <(

Spermatogonia (B) m

I Differentiation I

Lumen

After meiosis, haploid spherical spermatids differentiate into spermatozoa. Meiosis and differentiation take place in the adluminal compartment. Notice that each generation of cells is attached by intercellular cyto- plasmic bridges. Thus, each generation divides synchronously in cohorts. Some cells (black) degenerate during the process. Numbers indicate the theoretical number of cells generated by each division.

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206 Spermatogenesis

The goals of spermatogenesis are to: • provide a continual supply of male gametes (up to decades) through stem cell renewal

• provide genetic diversity

• provide billions of sperm each day (domestic animals) to maxi- mize reproduction by both natural service and artificial insemination

• provide an immunologically privileged site where developing germ cells are not destroyed by the immune system

Figure 10-4. Scanning Electron Micrograph of Testicular Parenchyma in the Stallion

(Courtesy of Dr. Larry Texas The A:nerican Society for Reproductive Med1cme. Fertil. and Stenl., 1978. 29.208-215)

Spermatogenesis 207

Spermatogenesis =proliferation + meiosis + differentiation

Spermatogenesis is the Process of Producing Spermatozoa

nia to revert to stem cells (spetmatogonial stem cells) providing continual renewal of these stem cells from which new spermatogonia can develop.

I Proliferation I

Basement membrane

Spermatogonia (A2) D Spermatogonia (A3) IJ Number of divisions depends on

species

Spermatogonia ( I ) IJI <(

Spermatogonia (B) m

I Differentiation I

Lumen

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208 Spermatogenesis

undergoes a remarkable transformation that results in the production of a fully differentiated, highly special- ized spermatozoon containing a head (nuclear material), a flagellum including a midpiece (with a mitochondrial helix) and a principal piece.

The most immature genn cells (spem1atogonia) are located at the periphe1y of a seminiferous tubule near the basement membrane. As these germ cells prolifer- ate, they move toward the lumen. The cell types in the seminiferous epithelium are illustrated in Figure 10-5. Developing germ cells are connected by intercellular bridges. Groups of spem1atogonia, spermatocytes or spenn atids are connected by intercellular bridges, so that the cytoplasm of an entire cohort (groups of cells of the same type) is interconnected. The exact number of genn cells that are interconnected is not !mown, but might approach 50. The significance of these intercel- lular bridges is not fully understood. However, they undoubtedly provide communication between cells that contributes to synchronized development of a cohort.

Proliferation Generates Spermatogonia That are Committed to Become More Advanced

Cell Types

The most primitive cells encountered in the seminiferous epithelium are the spemmtogonia. These specialized diploid (2N chromosomal content) cells are located in the basal compartment of the semi- niferous epithelium. Spermatogonia undergo several mitotic divisions with the last division resulting in primary spermatocytes (See Figure I 0-5). There are three types of spermatogonia: A-spermatogonia, !- spermatogonia (intem1ediate) and B-spermatogonia. A-spemmtogonia undergo several mitotic divisions in which they progress mitotically from A, through A4• A pool of stem cells is also maintained so that the process can continue indefinitely. Stem cells divide mitotically to provide a continual source of A-spermatogonia al- lowing spermatogenesis to continue without interrup- tion for years. The mechanism for the renewal of stem cells is not understood.

Meiotic Divisions Produce Haploid Spermatids

During spermatogenesis the number of chro- mosomes in the gamete is reduced to the haploid state. This is accomplished by meiosis. The mitotic divisions of 8-spennatogonia result in the fonnation of primmy spermatocytes . These primary spermatocytes imme- diately enter the first meiotic prophase. As you will recall fi·om your previous courses, the meiotic prophase consists of five stages: preleptotene, leptotene, zygo-

tene, pachytene and diplotene. Each of these stages represents a different step in the progression of DNA synthesis and replication. Primary spermatocytes must progress through these five steps before the first meiotic division can occur. The important event of the prelepto- tene phase is complete DNA replication forming tetrads without separation. These tetrads then fuse at random points known as chiasmata and crossing-over ofDNA material later takes place. The term "crossing-over" refers to segments of one chromosome crossing-over to a homologous chromosome when the chromatids separate. Crossing-over results in a random assort- ment of different segments of each clu·omosome. Thus, prophase of the first meiotic division insures genetic heterogeneity and that each secondary spem1atocyte and subsequently each spermatid will be genetically unique. Prophase of the first meiotic division is a relatively long process. In fact, the lifespan of the pri- maiy spermatocyte is the longest of all germ cell types found in the seminiferous epithelium. For example, in the bull the lifespan of the primmy spermatocyte is 18 to 19 days. The total duration of spennatogenesis in bulls is 61 days. Thus, prophase of the first meiotic division (primary spem1atocyte) is about 30% of the time required for the entire spermatogenic process.

The secondary spermatocyte resulting from the first meiotic division of a primary spermatocyte is short-lived. It exists for only l.l to 1.7 days depending on the species. The secondary spennatocyte rapidly undergoes the second meiotic division, resulting in haploid spherical spennatids.

Differentiation Produces a Highly Sophisticated, Self-Propelled Package

of Enzymes and DNA

The role of a spennatozoon is to deliver the male 's genetic material to an oocyte during fertilization. To form cells that are capable of fertilization, spherical spermatids undergo a series of changes in which the nucleus becomes highly condensed, the acrosome is formed and the cell becomes potentially motile. The abi lity to swim (motility) requires the development of a flagellum and a metabolic " powerplant" known as the mitochondrial helix.

Differentiation consists ofthe: • Golgi phase • cap phase • acrosomal phase • maturation phase

Spermatogenesis 209

Figure 10-6. The Golgi Phase of Spermatid Differentiation

0 0 e

The newly formed sper- Small vesicles of the Golgi Vesicle fusion continues until matid is almost perfectly fuse, giving rise to larger a large acrosomic vesicle is spherical and has a well secretory granules called formed containing a dense developed Golgi appa- pro-acrosomic granules. acrosomic granule. The proxi- ratus. The centrioles start to mi- mal centriole (PC) gives rise

grate to a position beneath to the attachment point of the the nucleus that is opposite tail . The distal centriole (DC) the acrosomic vesicle. gives rise to the developing

The Golgi phase = acrosomic vesicle formation

The Golgi phase is characterized by the first steps in the development of the acrosome. The newly fom1ed spermatid contains a large, highly-developed Golgi apparatus located near the nucleus that consists of many small vesicles (See Figure 1 0-6). The Golgi apparatus is not unique to the spermatid, but is the intracellular "packaging" system in a ll secretory cells. In a spermatid, the Golgi will give rise to an important subcellular organelle known as the acrosome. First, proacrosomic vesicles are formed and these fuse, gen- erating a larger vesicle that resides on one side of the nucleus. This vesicle is called the acrosomic vesicle and contains a dense acrosomic granule (See Figure I 0-6). Smaller Golgi vesicles are continually added to the larger vesicle increasing its size.

While the acrosomic vesicle is being fom1ed, the centrioles migrate from the cytoplasm to the base of the nucleus (See Figure 1 0-6). The proximal centriole will give ri se to an implantation apparatus that allows the flagellum to be anchored to the nucleus (See Figure I 0-9). The distal centriole gives rise to the develop- ing axoneme. The axoneme is the central portion of a flagellum, in this case the sperm tail.

axoneme (central portion of the tail) inside the cytoplasm of the spermatid.

The cap phase = acrosomic vesicle spreading over the nucleus

During the cap phase the acrosome fonns a distinct, easily recognized cap over the anterior portion of the nucleus (See Figure I 0-7). The Golgi now has performed its function by packaging the acrosomal con- tents and membranes and moves away from the nucleus toward the caudal end of the spem1atid and eventually disappears. The primitive flagellum (tail), fom1ed fi·om the distal centriole, begins to project from the spem1atid toward the lumen ofthe seminiferous tubule.

The acrosomal phase =nuclear and cytoplasmic elongation

During the acrosomal phase the acrosome continues to spread until it covers about two-thirds of the anterior nucleus (See Figures 10-7 and I 0-8). The nucleus begins to elongate. A unique system of mi- crotubules known as the manchette develops near the area of the posterior nucleus. Portions of the manchette attach to the region of the nucleus just posterior to the

101

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208 Spermatogenesis

Proliferation Generates Spermatogonia That are Committed to Become More Advanced

Cell Types

Meiotic Divisions Produce Haploid Spermatids

Differentiation Produces a Highly Sophisticated, Self-Propelled Package

of Enzymes and DNA

Differentiation consists ofthe: • Golgi phase • cap phase • acrosomal phase • maturation phase

Spermatogenesis 209

Figure 10-6. The Golgi Phase of Spermatid Differentiation

0 0 e

grate to a position beneath to the attachment point of the the nucleus that is opposite tail . The distal centriole (DC) the acrosomic vesicle. gives rise to the developing

The Golgi phase = acrosomic vesicle formation

axoneme (central portion of the tail) inside the cytoplasm of the spermatid.

The cap phase = acrosomic vesicle spreading over the nucleus

The acrosomal phase =nuclear and cytoplasmic elongation

101

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21 0 Spermatogenesis

Figure 10-7. The Cap, Acrosomal and Maturation Phases of Spermatid Differentiation

The Cap Phase

The Acrosomal Phase

The Maturation Phase

II<=.,;,-- Posrnucleor ---MI C:!p

Acrosome

Head

Principle piece

A The Golgi migrates toward the caudal part of the cell. The distal centriole (DC) forms the axoneme (AX) or flagellum that projects away from the nucleus toward the lumen of the seminiferous tubule.

B The acrosomic vesicle flattens and begins to form a distinct cap consisting of an outer acrosomal membrane (OAM), an inner acrosomal membrane (lAM) and the acrosomal contents (enzymes).

A The spermatid nucleus begins to elongate and the acrosome eventually covers the majority of the anterior nucleus. The man- chette forms in the region of the caudal half of the nucleus and extends down toward the developing flagellum.

B The neck and the annulus are formed and the later will become the juncture between the middle piece and the princi- pal piece. Notice that all components of the developing spermatid are completely surrounded by a plasma membrane. M = mitochondria.

AandB Mitochondria form a spiral assembly around the flagellum that defines the middle piece. The postnuclear cap is formed from the manchette microtubules. The annulus forms the juncture between the middle piece and the principal piece.

Figure 10-8. The Head of the Bovine Spermatozoon

(Courtesy of Dr. R.G. Saacke, Virginia Polytechnic Institute and State University wi th permission from

John R. Wiley and Sons, Inc. Am. J. Anat. 115:143)

Plasma me mbrane

Postnuclear cap

acrosome (See Figure I 0-7). Some of the microtu- bules of the manchette w ill become the postnuclear cap. During the acrosomal phase, spem1atids become deeply embedded in Sertoli cells with their tails pro- truding toward the lumen of the seminiferous tubule (See Figure 10-4).

The maturation phase =final assembly that forms a spermatozoon

During the maturation phase microtubules of the manchette direct the fonnation of the postnuclear cap. Mitochondria migrate toward and cluster around the flagellum in the region posterior to the nucleus. Mitochondria are quickly assembled around the flagel- lum from the base of the nucleus to the anterior one- third of the tail. They are assembled in a spiral fashion (See Figure 10-9) and fom1 the middle piece in fiilly differentiated spermatozoa. Dense outer fibers of the flagellum and the fibrous sheath are produced and final assembly is complete. It should be emphasized that, as in any cell, the entire spermatozoon is covered with a plasma membrane. Integrity of the plasma membrane is required for the survival and function of spem1atozoa as you w ill see later in the chapter.

Spermatogenesis 211

Spermatozoa = head + tail Head= nucleus +acrosome +post-

nuclear cap

Tail = middle piefe +principal piece + tenmnal p1ece

Finally, release of spermatozoa from the Sertoli cells into the lumen of the seminiferous tubule occurs. This release is referred to as spermiation and is analo- gous to ovulation in the female, except that spermiation occurs continuously throughout the testis.

The head of a mammalian spermatozoon has a shape characteristic for each species. In domestic mam- mals the nucleus is oval and fl attened and is surrounded by a nuclear membrane. The chromatin is compacted and is almost inert because it is highly keratinized. Ke- ratinoid proteins (hair, claws, hoofs and feathers) have a high degree of disulfide cross- linking and are quite insoluble. During spenniogenesis, nuclear histones of the haploid spem1 nucleus are replaced by protamines. Protamines are small, arginine-rich nuclear proteins thought to be essential for DNA condensation. The sufihydryl groups of protamines fonn d isulfide bonds. These bonds are the basis for nuclear condensation that results in a highly compact, stable nucleus that forms the spenn head. At this point in spenniogenesis, transcrip- tion and translation stops because the "transcriptional machinery" can no longer access nuclear DNA. Most of the "translational machinery" has been partitioned and lost within the residual cytoplasm ofthe spennatid. The DNA within the spenn head remains fundamentally inert unti l the time of fertilization. The inert nature of the DNA is thought to be a mechanism to prevent damage to the DNA between spem1iation and ferti lization. At fertilization, the process is reversed because the disulfide cross-links within the sperm nucleus are reduced by glu- tathione in the cytoplasm of the oocyte. The protamines are replaced with histones from the oocyte cytoplasm resulting in nuclear decondensation and fonnation of the male pronucleus (See Figure 12-8). Thus, the process of nuclear condensation, characterized by a high degree of keratinization and DN A stability is reversed only after the spem1 enters the oocyte cytoplasm.

The anterior two-thirds of the nucleus is cov- ered by the acrosome. The acrosome is a membrane- bound lysosome that contains hydrolytic enzymes. These enzymes, acrosin, hyaluronidase, zona lysin, esterases and acid hydro lases, are required for penetra- tion ofthe cellular investments and the zona pellucida of the ovulated oocyte. During ferti lization the acrosome undergoes an ordered, highly specialized exocytosis, known as the acrosome reaction, that allows release of the enzymes that are packaged in it to digest or penetrate

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21 0 Spermatogenesis

Figure 10-7. The Cap, Acrosomal and Maturation Phases of Spermatid Differentiation

The Cap Phase

The Acrosomal Phase

The Maturation Phase

II<=.,;,-- Posrnucleor ---MI C:!p

Acrosome

Head

Principle piece

A The Golgi migrates toward the caudal part of the cell. The distal centriole (DC) forms the axoneme (AX) or flagellum that projects away from the nucleus toward the lumen of the seminiferous tubule.

B The acrosomic vesicle flattens and begins to form a distinct cap consisting of an outer acrosomal membrane (OAM), an inner acrosomal membrane (lAM) and the acrosomal contents (enzymes).

Figure 10-8. The Head of the Bovine Spermatozoon

(Courtesy of Dr. R.G. Saacke, Virginia Polytechnic Institute and State University wi th permission from

John R. Wiley and Sons, Inc. Am. J. Anat. 115:143)

Plasma me mbrane

Postnuclear cap

The maturation phase =final assembly that forms a spermatozoon

Spermatogenesis 211

Spermatozoa = head + tail Head= nucleus +acrosome +post-

nuclear cap

Tail = middle piefe +principal piece + tenmnal p1ece

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212 Spermatogenesis

Figure 10-9. The Tail of the Bovine . . (Courtesy of Dr. R.G. Saacke, Virginia Polytechnic Institute and State Umvers1ty w1th perm1ss1on

from John R. Wiley and Sons, Inc. Am. J. Anat. 115:163)

I \

Capitulum

Coarse outer fibers

Mitochondrial helix

Longitudinal elements

Axoneme (9 dublets + 2 central

fibers)

Coarse outer fibers

Fibrous helix

piece

Principal piece

• • .J

the zona pellucida. These reactions will be described in more detail in Chapter 12. Acrosomal morphology var- ies among species, but in the boar, ram, bull and stallion the acrosome is similar to that shown in Figure 10-8 . The membrane component posterior to the acrosome is the postnuclear cap.

The sperm tail is a self-powered flagellum.

The tai l is composed of the capitulum, the middle piece, the principal piece and the terminal piece. The capitulum fits into the implantation socket, a depression in the posterior nucleus. The anterior por- tion of the tail consists of laminated columns that give the neck region flexibility when it becomes motile, so the tail can move laterally from side-to-side during the flagellar beat. The axonemal component of the tail originates fi·om the distal centriole and is composed of 9 pairs of microhibules that are ananged radially around two central filaments. Surrounding this 9+9+2 arrangement ofmicrotubules are 9 coarse fibers that are unique to the flagellum of spennatozoa. This arrange- ment ofhibules in the tail ofspern1atozoa is illustrated in Figure 10-9.

The mitochondrial sheath is arranged in a heli- cal pattem (See Figure 1 0-9) around the outer coarse fibers of the tail and contributes to the middle piece. The annulus demarcates the j uncture between the middle piece and the principal piece. The principal piece makes up the majority of the tail and continues almost to the end of the flagellum, where only the microh1bules end in the terminal piece.

Spermatozoa are Released Continually into the Lumen of the Seminiferous Tubules

One of the major differences between gamete production in the female and the male is that the fe- male's gamete supply is produced entirely before birth. After puberty, she begins to produce oocytes that will undergo meiosis and ovulate every 3-4 weeks. Thus, maturation, meiosis and release of female gametes is pulsatile. In contrast, the male produces gametes continually and unifom1ly throughout his reproductive lifespan. An exception to this is the seasonal breeder that produces spermatozoa during the breeding season only. Understanding the mechanisms responsible for the continual production of spermatozoa by the semi- niferous epithelium represents a major challenge for sh1dents of reproductive physiology.

Appreciating the spermatogenic process is necessary for a complete understanding of reproductive physiology. But the importance of this understanding

Spermatogenesis 213

goes beyond the academic. From a clinical perspective, evaluation of sperm numbers in the ejaculate does not always accurately reflect nonnal or abnonnal spermato- genesis. Therefore, the fate of males being evaluated is often fraught with error and thus bad decisions are made. One needs to understand that there is a 2 to 4 week delay before the effects of deleterious events (heat stress, shipping, fever, exposure to certain toxins) can be observed by monitoring changes in ejaculated spenn. Furthermore, 6 to 12 weeks are required before restora- tion of normal spermatogenesis can be accomplished after these events. Therefore, clinical interpretations of ejaculate characteristics requires specific knowledge of the timing of spermatogenesis in the species being evaluated. Seasonal spermatogenesis requires that the genninal epithelium "him-on" and "tum-off" as a function of environmental influences. More and more emphasis is being placed on "saving and managing" endangered species. For these efforts to be successful, the timing of spermatogenesis and sperm producing potential must be understood so that sufficient male gametes are avai lable for reproductive manipulation (artificial insemination, in vitro fertil ization, etc.). As of yet, a practical, cost-effective contraceptive is not available for men. We need to leam how to temporarily "tum-off" and later "turn-on" spennatogenesis without altering the behavior of the male. Our ability to manipu- late male gamete production will play a major part in the abi lity to manipulate reproduction in the future.

In order to comprehend the cycle of the seminiferous epithelium you must first understand:

• cellular generations • stages of the cycle • duration of one cycle • how the cycle is repeated

The cycle of the seminiferous epithelium is the progression through a complete series of cellular associations (stages) at one location along a seminifer- ous tubule. The time required for this progression is the duration of the cycle of the seminiferous epithelium and is unique for each species.

Germ cell generations are cells of the same type located at one site within the

seminiferous epithelium.

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212 Spermatogenesis

Figure 10-9. The Tail of the Bovine . . (Courtesy of Dr. R.G. Saacke, Virginia Polytechnic Institute and State Umvers1ty w1th perm1ss1on

from John R. Wiley and Sons, Inc. Am. J. Anat. 115:163)

I \

Capitulum

Coarse outer fibers

Mitochondrial helix

Longitudinal elements

Axoneme (9 dublets + 2 central

fibers)

Coarse outer fibers

Fibrous helix

piece

Principal piece

• • .J

The sperm tail is a self-powered flagellum.

Spermatozoa are Released Continually into the Lumen of the Seminiferous Tubules

Appreciating the spermatogenic process is necessary for a complete understanding of reproductive physiology. But the importance of this understanding

Spermatogenesis 213

In order to comprehend the cycle of the seminiferous epithelium you must first understand:

• cellular generations • stages of the cycle • duration of one cycle • how the cycle is repeated

Germ cell generations are cells of the same type located at one site within the

seminiferous epithelium.

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214 Spermatogenesis

Within any given microscopic cross-section of a seminiferous tubule, one can observe four or five concentric "layers" of germ cells. Cells in each layer comprise a generation. A generation is a cohort of cells that develops as a synchronous group. Each generation of cells (each concentric layer) has a similar appear- ance and function. Cross-sections along the length of a seminiferous tubule will have a different appear- ance but the entire cross-section at a given location will usually appear similar. For example, while view- ing cross-section I (stage I) in Figure l 0-10, you will observe four generations of genu cells. Each generation will give rise to a succeeding, more advanced genera- tion. Observe in Figure 10-10 that there is a generation of A -spermatogonia near the basement membrane in the

section of the h1bule labeled Stage I. Just above the A -spermatogonia is a young generation of primary sper- matocytes. Above it lies a third generation consisting of more mahlre prima1y spermatocytes. Finally, near the lumen, is a fomth generation of cells. This generation consists of spherical immarure spennatids. Remember that the more immah1re cell types are generally located near the basement membrane (basal compartment) and the more advanced cell types reside in the adluminal compartment.

In cross-section IV (stage IV) of Figure 10- 10, there are five generations of genn cells. You will observe a generation of A-spermatogonia, one genera- tion of intermediate spermatogonia, one generation of primary spermatocytes, one generation of secondary

Figure 10-10. Associations of Developing Germ Cells That Represent Various Stages of the Cycle of the Seminiferous Epithelium

VIII ll ill IV V VI VII VITI

Stage I

A stage I tubule consists of 1 generation of A- spermatogonia, 2 generations of primary sperma- tocytes ( 1 o cyte) and 1 generation of immature sper- matids ('Tid).

\..

At any given cross-sectioned loca- tion along a seminiferous tubule, one can observe different stages of the cycle of the seminiferous epithelium. In this example, we see three stages (I, IV, and VIII).

'gonia = spermatogonium 1 o cyte = primary spermatocyte 2° cyte = secondary spermatocyte 'Tid = immature spermatid 'Tid-m =mature spermatid

Stage IV Stage VIII

A stage IV tubule con- sists of 2 generations of spermatogonia (A+I), 1 generation of primary spermatocytes (1 o cyte ), 1 generation of second- ary spermatocytes (2° cyte) and 1 generation of immature spermatids ('Tid).

A stage VIII tubule consists of 2 genera- tions of spermatogonia (A+B), 1 genera- tion of primary spermatocytes (1° cyte) and 2 generations of spermatids ('Tid). The young generation of spermatids ('Tid) have formed only a few days earlier and are quite immature. The second generation of spermatids are mature ('Tid-m) and are about to be released into the lumen.

Spermatogenesis 215

Figure 10-11. Cycle of the Seminiferous Epithelium in the Bull (Modified from Amann, R.P. Am. J. Anat. 110:69)

Spermiation

Lumenl 61.days

5 ---- ---- c 0

•.t; f C1) c C1)

C) --C1) u

2 ...... __ _ --- --

----

---- "(- -- --

VIII

13.5

• Horizontal axis - Stage of cycle and days spent in each stage. • Vertical axis = Cell generations in each stage i.e. type of cell seen from the basal level to the luminal

level within a cross section of a seminiferous tubule. • Horizontal line= Developmental pathway from spermatogonia to spermatozoa (61 days). • The release of spermatozoa from the Sertoli cells occurs in stage VIII and is called spermiation.

It occurs 61 days after A-spermatogonia are formed at the beginning of Stage Ill. Ocell division (mitotic for 'gonia, meiotic for primary and secondary 'cytes). • In the bull, it takes about 4.5 cycles of the seminiferous epithelium to complete spermatogenesis

(4.5 cycles x 13.5 days/cycle = 61 days).

spennatocytes and one generation of spermatids. The spermatids in stage IV are elongated and, thus are more advanced than the spemmtids in stage I.

In cross-section VIII (stage VIII), there are also five generations of gem1 cells. Observe two generations of spermatogonia (one generation of A and one genera- tion of B-spermatogonia), one generation of primary spem1atocytes and two generations of spermatids. One generation of spermatids is rather immahlre and spheii- cal, while the more advanced generation consists of mature spemmtids ready for release from Sertoli cells into the lumen of the seminiferous tubule.

At one instance in time, three cross-sections at d ifferent locations along the seminiferous tubule show different generations of cells . Cells in each section are actively engaged in spennatogenesis, but only one cross- section (VIII) is ready to release spermatozoa into the lumen. Thus, along the length of any seminiferous tu- bule there are only certain zones (cross-sections) where spermatozoa are released at any given point in time. All other zones or stages are preparing to release spenna- tozoa, but the cells in those zones have not reached the appropriate stage of maturity for spermiation to occur.

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214 Spermatogenesis

Figure 10-10. Associations of Developing Germ Cells That Represent Various Stages of the Cycle of the Seminiferous Epithelium

VIII ll ill IV V VI VII VITI

Stage I

A stage I tubule consists of 1 generation of A- spermatogonia, 2 generations of primary sperma- tocytes ( 1 o cyte) and 1 generation of immature sper- matids ('Tid).

\..

'gonia = spermatogonium 1 o cyte = primary spermatocyte 2° cyte = secondary spermatocyte 'Tid = immature spermatid 'Tid-m =mature spermatid

Stage IV Stage VIII

Spermatogenesis 215

Figure 10-11. Cycle of the Seminiferous Epithelium in the Bull (Modified from Amann, R.P. Am. J. Anat. 110:69)

Spermiation

Lumenl 61.days

5 ---- ---- c 0

•.t; f C1) c C1)

C) --C1) u

2 ...... __ _ --- --

----

---- "(- -- --

VIII

13.5

• Horizontal axis - Stage of cycle and days spent in each stage. • Vertical axis = Cell generations in each stage i.e. type of cell seen from the basal level to the luminal

(4.5 cycles x 13.5 days/cycle = 61 days).

spennatocytes and one generation of spermatids. The spermatids in stage IV are elongated and, thus are more advanced than the spemmtids in stage I.

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216 Spermatogenesis

= specific cellular associations duration= time required for completion of one stage (cell association)

Cvcle =progression through sequence of all stages

Cvcle dumtion = time required to complete one cycle

Figure 10-12. The Cycle of Seminiferous Epithelium is Analogous to a University

(Modified from Johnson, 1991)

Every year, freshmen (spermatogonia) enter and seniors (spermatozoa) graduate. How- ever four years are required for a freshmen to through the various classes become a graduating senior. Each class 1s analogous to a generation of germ cells found in the seminiferous epithelium.

Senior I

('tid) I ... ______________

Junior (Secondary 'cyte) I

..............................

Graduation (Spermiation)

"""4 years

Sophomore (Primary 'cyte) ® ..... 0

Freshman ('gonia)

, ______________ _

Fall semester

------------- ....

Spring semester

I year

\ I ,

"Flunk-out"

Stages of the cycle are arbitrarily defined cellular associations that transition one to

the next at predictable intervals.

As previously explained, sections or zones along a seminiferous tubule contain different cellular associations. These cellular associations, or stages of the cycle of the seminifet·ous epithelium, have been defined arbitrarily by researchers who have made thou- sands and thousands of observations of the seminiferous epithelium using light microscopy.

If you were to microscopically scan a number of tubules in the testicular parenchyma, you would see tubule cross-sections that contain exactly the same cell types and relationships as other tubules. In fact, with enough observation you would begin to encounter dif- ferent cross-sections with definable cellular composi- tions at predictable frequencies. For the purposes of this text, we will describe eight stages in the cycle of the seminiferous epithelium, even though other schemes are available with as many as 14 stages.

Figure 10-11 illustrates the cellular composi- tion of each stage of the seminiferous epithelium. For example, stage I contains one generation of A-sper- matogonia, two generations of primary spermatocytes and one generation of spennatids. By scanning from the basement membrane (bottom of diagram) toward the lumen, you can quickly detennine which cell types are present at each of the eight stages.

Lifespan of cells and duration oftlte cycle vary among species.

The entire progression of one cycle of the seminiferous epithelium from stage I through stage VIII requires 13.5 days in the bull (for other species see Table 1 0-1 ). That is, if you could observe one cross-section of a seminiferous tubule continually, starting at the beginning of stage I, it would require 13.5 days before you would observe spem1iation (the end of stage VIII). After spem1iation (end of stage VIII), the cross section you were observing would again have the same cellular association as it did on the day you s tarted watching (stage 1). Thus, one cycle of the seminiferous epithe- lium would have been completed.

The complete process ofspennatogenesis from A-spennatogonia to the fonnation of fully differentiated spermatozoa takes 61 days in the bull. During the 61 days, cells at a given area ofthe seminiferous epithelium proceed through 4.5 cycles of the seminiferous epithe- lium (13 .5 days/cycle X 4.5 cycles = 61 days).

Spermatogenesis 217

This process is analogous to a traditional uni- versity. Every year a new class of freshmen enters the university in the fall. These freshmen are analogous to committed A-spemmtogonia entering the spemlato- genic pathway. The freshmen (A-spermatogonia) un- dergo noticeable changes during the first year, and after one year they become sophomores. Sophomores are analogous to primaty spem1atocytes. The sophomores (primary spermatocytes) also undergo maturational changes and become juniors (secondary spermatocytes; although they actually are short-lived). Finally, they become seniors (spennatids) and graduate after four years (See Figure 10-12).

The cycle of the seminiferous epithelium is almost identical in concept to the university situation, except the school year is only 13.5 days (1 cycle of the seminiferous epithelium in the bull). Every 13.5 days a new generation of freshmen (A-spermatogonia) enter and a generation of seniors (spermatids) gradu- ate. Graduation by the seniors is analogous to sperm- iation. Remember, it takes four years to graduate from the university. Similarly, it takes 4.5 cycles for an A-spermatogonium (freshman) to become a fully dif- ferentiated spermatozoon (senior). A major difference between the university example and the actual cycle of the seminiferous epithelium is that the germinal elements have different lifespans. For example, a primary spem1atocyte exists for about 21 days while a secondary spermatocyte exists for only 1. 7 days in the bull. In the university, freshmen, sophomores, juniors and seniors have similar lifespans (assuming a basal academic perfonnance ).

There is another major difference between the university analogy and what actually takes place in the genninal epithelium. Spennatogonia (freshmen), primary (sophomores) and secondary (juniors) spenna- tocytes all divide and generate many spennatids. For example each incoming freshmen (A-gonia) could theo- retically produce 256 seniors (spennatids). Obviously, such multiplication does not take place with university students. In the university, a significant proportion of entering freshmen "flunk-out" and never graduate, so there are always more freshmen than graduating seniors. Similarly, during spennatogenesis many proliferating spennatogonia die and never become primary spennato- cytes. Therefore, the numberofprimruy spermatocytes generated per conm1itted A-spem1atogonium is closer to 20-30 than the theoretical 64 as depicted in Figure I 0-5. There also is death of primary spem1atocytes, although most spherical spennatids do form a sper- matozoon. In contrast from a university where each student can choose their pace throughout the years, to amass 120 credits, in the testis of a given species, the pace through spemmtogenesis is essentially identical and is not affected by environment.

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216 Spermatogenesis

= specific cellular associations duration= time required for completion of one stage (cell association)

Cvcle =progression through sequence of all stages

Cvcle dumtion = time required to complete one cycle

Figure 10-12. The Cycle of Seminiferous Epithelium is Analogous to a University

(Modified from Johnson, 1991)

Senior I

('tid) I ... ______________

Junior (Secondary 'cyte) I

..............................

Graduation (Spermiation)

"""4 years

Sophomore (Primary 'cyte) ® ..... 0

Freshman ('gonia)

, ______________ _

Fall semester

------------- ....

Spring semester

I year

\ I ,

"Flunk-out"

Stages of the cycle are arbitrarily defined cellular associations that transition one to

the next at predictable intervals.

Lifespan of cells and duration oftlte cycle vary among species.

Spermatogenesis 217

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218 Spermatogenesis

Table 10-1. Duration of the Stages of the Cycle of the Seminiferous Epithelium in Various Species

Stage Bull Ram Boar Stallion Rabbit I 4.2 2.2 1.1 2.0 3.1 II 1.2 1.1 1.4 1.8 1.5 Ill 2.7 1.9 0.4 0.4 0.8 IV 1.7 1.1 1.2 1.9 1.2 v 0.2 0.4 0.8 0.9 0.5 VI 0.8 1.3 1.6 1.7 1.7 VII 1.1 1.1 1.0 1.6 1.3 VIII 1.6 1.0 0.8 1.9 0.9

TOTAL A 13.5 10.1 8.3 12.2 11 .0 SPERMATOGENESIS8 61 47 39 55 48

ATotal days required for 1 cycle of the seminiferous epithelium BApproximate days to complete spermatogenesis (sperm.atogonia to spermatozoa)

The spermatogenic wave is the sequential ordering of stages along the length of the

seminiferous tubule.

The duration of each stage of the cycle of the seminiferous epithelium varies with species, as does the length of the cycle of the seminiferous epithelium. Variations in stage, cycle length and total time required for spemmtogenesis are presented in Table 1 0-l.

The spermatogenic wave refers to the differ- ences at any given instant in time along the length of the seminiferous tubule. Imagine that you could run down the lumen of the seminiferous tubule. As you run down the tubule, you will encounter zones that are near spem1iation (stage VIII). The distance between these spermiation sites is relatively constant. During the wave, each stage of the seminiferous epithelium transitions to a successively more advanced stage. For example, a stage I tubule will later become a stage II and stage II will later become a III and so on. Thus, the site of spermiation along the tubule is constantly changing, creating a "wave" ofspenn release down the length of the tubule. This "wave" is like the wave con- ducted by football fans in a stadium. When the fans stand up, they mimic spenniation. They sit back down and don't stand up again until it's their tum again. The time spent sitting (stages I-VII) is much longer than the time spent standing. As the wave in the stadium continues, repeated standing and sitting takes place at a relatively constant rate. So does spenniation. The physiologic importance of the spermatogenic wave is to provide a relatively constant supply of spem1atozoa to the epididymis, creating a pool for ejaculation.

Figure 10-13. Scrotal Circumference Measurements are Good Indicators of Sperm

Producing Ability (Photograph courtesy of Select Sires, Inc.

Plain City, Ohio, www.selectsires.com)

Accurate scrotal circumference measurements require that both testicles be pushed ventrally by applying pressure to the spermatic cord. A specially designed tape is then placed around the scrotum at its widest point and a measure- ment is taken (in this case, 40cm).

Spermatogenesis 219

Table 10-2. Testicular Characteristics and Sperm Production Estimates of Sexually Mature Mammals SBecies Gross weight of SBerm Broduced Daily

Baired testes Ber gram of SBermatozoal (grams) testicular Barenchyma Broduction

Beef Bull 650 11x1 06 6x109 Boar 750 23x106 16x109 Cat 21 16x106 32x1 06 Dairy Bull 725 12x106 7.5x109 Dog (16 kg body weight) 31 17x106 0.50x109 Man 35 4x106 0.13x109 Rabbit 6 25x106 0.20x109 Ram* 550 21x106 10x109 Rooster*** 25 100x106 2.5x109 Stallion** 340 16x1 06 5x1 09

*in breeding season (shortening-day length), **in breeding season (increasing day l!:mgth), · ***varies greatly with management and strain

Daily sperm production (DSP) is defined as the total number of spennatozoa produced per day by both testicles of the male. Accurate measurement of DSP requires removal of all or a portion of the testicle and thus, DSP cannot be measured using non- invasive techniques . However, noninvasive measures such as total number of spermatozoa ejaculated into an artificial vagina with daily ejaculations for 2-3 weeks gives a good estimate of DSP. Interspecies variation in testicular weights, sperm produced per gram of testicular parenchyma ·and daily sperm production is presented in Table 10-2. The number of spermatozoa produced per day per gram of testicular parenchyma is referred to as efficiency of sperm production. Daily sperm production is dependent, at least in part, on the number of Sertoli cells populating the testes. For example, the higher the number of Sertoli cells, the higher the spem1atozoal production rates. Numbers of Sertoli cells also have been positively correlated with spermatogonial and spermatid numbers.

Testicular Size is a Good Estimator of Sperm Producing Ability

To detennine a given male 's sperm producing capability, it is necessary to collect ejaculates from the animal for a period of time. This enables one to ac- curately estimate how many spem1atozoa the animal can produce per unit time. If collection of semen is not possible, a good estimate ofspenn producing capability can be made by measuring the circumference of both testicles (See Figure 1 0- I 3 ). The greater the testicular circumference, the greater the sperm producing capabil-

ity, in other words, "the bigger the factory, the greater the output." Because of the non-pendular scrotum in the boar and stallion, scrotal width or length is used as the measurement.

Assuming that a male can develop an erect penis, mount and ejaculate in the female, his potential fertility is determined by:

• his sperm producing ability

• the viability of his spermatozoa

• the number of morphologically abnor- mal spermatozoa that he ejaculates

• the number of functionally normal spermatozoa that he ejaculates

Spermatozoal Viability is Judged by Evaluating Motility

Even though a male can produce large quanti- ties of spem1atozoa, it is important that these spem1 are alive and highly motile. M oti lity is generally de- scribed as the ability of spe1m to swim progressively forward. Moti lity is the most commonly used assess- ment of viability. It is expressed as an estimate of the percentage of sperm that are swimming in a linear fashion with in a given environment as determined mi- croscopically. Unfortunately, the relationship between percentage of motile sperm and fertility is not a good one. However, if few spennatozoa within a series of

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218 Spermatogenesis

Table 10-1. Duration of the Stages of the Cycle of the Seminiferous Epithelium in Various Species

TOTAL A 13.5 10.1 8.3 12.2 11 .0 SPERMATOGENESIS8 61 47 39 55 48

ATotal days required for 1 cycle of the seminiferous epithelium BApproximate days to complete spermatogenesis (sperm.atogonia to spermatozoa)

The spermatogenic wave is the sequential ordering of stages along the length of the

seminiferous tubule.

Figure 10-13. Scrotal Circumference Measurements are Good Indicators of Sperm

Producing Ability (Photograph courtesy of Select Sires, Inc.

Plain City, Ohio, www.selectsires.com)

Spermatogenesis 219

Table 10-2. Testicular Characteristics and Sperm Production Estimates of Sexually Mature Mammals SBecies Gross weight of SBerm Broduced Daily

Baired testes Ber gram of SBermatozoal (grams) testicular Barenchyma Broduction

*in breeding season (shortening-day length), **in breeding season (increasing day l!:mgth), · ***varies greatly with management and strain

Testicular Size is a Good Estimator of Sperm Producing Ability

ity, in other words, "the bigger the factory, the greater the output." Because of the non-pendular scrotum in the boar and stallion, scrotal width or length is used as the measurement.

Assuming that a male can develop an erect penis, mount and ejaculate in the female, his potential fertility is determined by:

• his sperm producing ability

• the viability of his spermatozoa

• the number of morphologically abnor- mal spermatozoa that he ejaculates

• the number of functionally normal spermatozoa that he ejaculates

Spermatozoal Viability is Judged by Evaluating Motility

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220 Spermatogenesis

Figure 10-14. Some Common Abnormalities in Bovine Sperm as Observed With Differential-Interference Contrast Microscopy

(Courtesy of R.G. Saacke, Virginia Polytechnic Institute and State University)

Head Abnormalities Crater Defect

(Nuclear Vacuoles) Tapered Heads Ruffled Acrosome Knobbed Acrosome

Tail Abnormalities Coiled Tail Double Midpiece Folded Tail Detached Head

Note: A vast amount of information is available for bulls because of intense scrutiny given to abnormal sperm by commercial AI organizations. For details on the incidence, causes and their effects on fertility of abnormal sperm shown here (and other types as well) see Barth and Oko, 1998 in the Key References section at the end of the chapter. Most descriptions in the bull apply to other mammals as well.

ejaculates are motile, the assumption can be made correctly that spenn in the ejaculate are not alive and therefore cannot fertilize the egg. There are many ways to tell if a spennatozoon is alive. These include oxygen consumption, exclusion of certain dyes by the plasma membrane (live-dead stains) and examination by flow cytometry. However, the simplest and most common is to determine if a cell moves forward in a progressive manner (motile) when examined at 37°C. Evaluating motility at temperatures below 3 7oc is not a good practice because motility stops at about l8°C. The use of a phase-contrast microscope (essential to clearly visualize spenn) and a heated stage (to allow sperm to display their potential to swim) is the most practical way to evaluate motility of sperm. Decisions about motility should never be based solely on one ejaculate.

There are Many Types of Abnormal Spermatozoa

As you might imagine, a process that poten- tially produces up to 20 billion spenn per day (over 200,000 per second) will have enors. These errors are expressed as abnormal spermatozoa some of which can be detected on the basis of abnomml shape. Mor- phologically abnom1al spem1 can be defined as any shape characteristic deviating from normal. Every ejaculate will contain between 5 and 15% abnormal sperm and these levels are generally considered ac- ceptable. Reduced fertility may result when morpho- logically abnormal sperm exceed 20% of sperm in the ejaculate. Some morphologic abnonnalities have a severe effect on fertility while others have little or no

effect. In general, morphologic abnormalities either originate in the testes because of faulty differentiation or in the epididymis because of faulty epididymal transit and/or maturation. The latter results in the presence of cytoplasmic droplets (See Chapter 3 ). Morphologically abnormal sperm of testicular origin are generally classi- fied as either head abnormalities or tail abnonnalities.

Potential fertil ity of the male can be related to the percentage of morphologically abnormal spenn within an ejaculate. Some common abnonnalities in bull spem1 are shown in Figure 10-14. Some abnor- malities are heritable and result in sterility. Males possessing these abnormalities should be eliminated from the gene pool.

Evaluation of the proportion of abnormal sperm in an ejaculate requires a microscope. For most laboratories, a phase-contrast microscope and a skilled observer will yield satisfactory diagnoses. For laboratories examining large numbers of ejaculates, a differential-interference contrast microscope is preferred because of the high resolution and the cellular detail generated with this optical system. A differential-interference contrast microscope transfonns gradients in intracellular density into an optical image that appears as a relief or an indentation in the cell. Thus, abnormalities of both the head and tai l can be observed and quantitated with a high degree of preci- sion. All ofthe micrographs presented in Figure 10-14 were generated with a differential-interference contrast microscope. A description of each type of abnormality that one can encounter within a series of ejaculates is beyond the scope of this text.

It must be recognized that morphologic ab- normalities represent only one characteristic among a myriad of possibilities for abnormal function. For example, abnormal nuclear composition (faulty DNA), abnormal biochemical composition, surface protein deficiency and faulty response to stimuli within the female tract represent only a few possibilities that may limit the function of spermatozoa.

Artificial Insemination is the Single Most Important Physiologic Technology Ever

Devised for Accelerating Genetic Improvement

The components of artificial insemination (AI) in the fact box, will be presented in the chapters that discuss the physiology of each process. For example, collection of semen involves behavioral issues requir- ing specific stimuli for mounting and ejaculation (See Chapter II). Preservation and extension of semen is an issue associated with providing an optimum in-vitro environment to preserve sperm viability (Current Chap- ter). Finally, insemination of the female delivers sperm

Spermatogenesis 221

to the female reproductive tract so that adequate num- bers are present and fertilization can be accomplished (See Chapter 12). Successful AI can be accomplished in any species provided the criteria below are met.

The majm· steps of artificial insemination are: • collection of semen from the male (See Chapter 11)

• preservation and extension of sperm (See Below)

• insemination of the female (See Chapter 12)

Artificial insemination is a common practice in some species. For example, over 7 million dairy cows and about 2 million beef cows are artificially inseminated annually in the United States. All turkey hens in commercial flocks (over 300 million) are artifi- cially inseminated because the toms have such a broad breast that they cannot mount and copulate. The use of AI in swine has exploded during the past 10 years. Approximately 85% of all female swine are artificially inseminated in the United States. This means that about 5 million females are artificially inseminated each year. Typically, each female receives about 2.3 inseminations per year in order to deliver two litters per year. This means that in the swine industry there are about 11.3 million artificial inseminations per year. This means that nearly 120 million pigs are s ired by artificial in- semination in the United States each year.

It should be emphasized that widespread ap- plication of artificial insemination allows for intense and relatively rapid genetic selection that significantly improves production efficiency in dairy, beef, poultry and swine. Improvements in animal efficiency result in a wide variety ofhighly affordable animal products to the consumer. Artificial insemination is also common for horses. In addition, many species in zoos have been artificially inseminated to avoid inbreeding and facili- tate reproduction in exotic and endangered species. In 2002 the first baby elephant was produced by artificial insemination at the National Zoo in Washington D.C. Artificial insemination is used routinely in assisted reproductive techniques in humans, allowing pregnan- cies to occur that otherwise would not be possible (See Chapter 16).

10]

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220 Spermatogenesis

Figure 10-14. Some Common Abnormalities in Bovine Sperm as Observed With Differential-Interference Contrast Microscopy

(Courtesy of R.G. Saacke, Virginia Polytechnic Institute and State University)

Head Abnormalities Crater Defect

(Nuclear Vacuoles) Tapered Heads Ruffled Acrosome Knobbed Acrosome

Tail Abnormalities Coiled Tail Double Midpiece Folded Tail Detached Head

There are Many Types of Abnormal Spermatozoa

Artificial Insemination is the Single Most Important Physiologic Technology Ever

Devised for Accelerating Genetic Improvement

Spermatogenesis 221

The majm· steps of artificial insemination are: • collection of semen from the male (See Chapter 11)

• preservation and extension of sperm (See Below)

• insemination of the female (See Chapter 12)

10]

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222 Spermatogenesis

Immediately after collection, the following information is needed:

• ejaculate volume

• concentration of spermatozoa in the ejaculate (sperm/mL ejaculate)

• percentage of motile sperm

In Vitro Preservation is Obligatory for Successful AI

After semen has been collected from the male, in vitro preservation of sperm for a period of time must be accomplished before successful delivery ofspenn to the female can take place. Preservation and dilution of sperm requires an environment that minimizes death of spem1. It also requires )mow ledge about the volume of the ejaculate, the concentration of sperm in the ejaculate and their motility.

Having the above information is necessary to determine the appropriate dilution rate of the spenn so multiple females can be inseminated with sperm from the same ejaculate. Where multiple females are to be inseminated, one must know the concentration ofspenn in the ejaculate so that each female can be inseminated with a threshold number (minimum number) of spenna- tozoa to maximize the probability of a pregnancy.

Evaluation of Semen is Needed Before Dilution

Immediately after collection of the ejaculate seminal evaluation is conducted. First, ejaculate vol- ume must be determined. Second, the percentage of sperm displaying progressive motility (swimming in a linear fashion) is estimated by viewing live smears at 37°C with a phase-contrast microscope. Third, the concentration of spermatozoa in the ejaculate is de- termined by comparing optical density of a standard dilution of neat semen with a reference standard. The greater the spenn concentration, the greater the optical density. The sperm concentration is determined from a standard curve where optical density is plotted against concentration.

The ejaculate volume and concentration of spennatozoa are important elements of seminal evalu- ation because the volume multiplied by the concentra- tion equals the total number of sperm in the ejaculate as shown in the equation below.

Total Sperm in Ejac. = Ejac. Vol. x Sperm/ml

Knowing the total number of sperm in the ejaculate enables the laboratory technician to determine how many insemination doses are potentially avai lable within each ejaculate.

A high percentage of motile sperm (60% or more) indicates good quality. An ejaculate containing few motile sperm (less than 50%) is a candidate for discard especially if sperm are to be frozen and later thawed to inseminate females.

Information for determining the number of insemination doses contained in a typical ejaculate for the bull is presented below. These calculations apply in principle to other species except that the values (vol- ume, concentration and motility) may vary significantly from spec ies-to-species and from male-to-male.

Ejaculate volume = 6 ml

Sperm concentration = 1.0 X I 09 sperm/ml (I billion)

Total sperm in ejaculate = 6 ml x I .Ox I 09sperm/m i = 6x I 09 (6 billion)

Progressive motility = 70%

Total motile sperm= 6.0xl09 x.7 = 4.2x I09 motile sperm/ejaculate

Desired concentration = 15x I 06/dose ( I insemination)

Number of doses= 4.2x I 09/15x I 06 = 280 doses

To determine the number of doses a single ejaculate will generate, one must divide the total number of sperm by the desired number of spenn in each dose. For example, the ejaculate illustrated above contains 4.2 billion motile sperm. If a dose of semen is intended to contain 15 million motile spenn ( 15x I 06) then we divide 4.2x I 09 sperm by 15x 1 06 sperm. By the computation in the box above, this ejaculate will produce 280 doses (units) of semen after di lution.

Good seminal extenders must:

• be isotonic • be good buffers • minimize cold damage ("cold shock'') • provide appropriate nutrients • prevent microbial growth • maintain viability • be relatively low in cost

Seminal Extenders Extend Both Sperm Viability and Numbers

After it has been determined that the ejaculate is of sufficient quali ty (volume,% motile spermatozoa and concentration of spermatozoa) the sperm must be preserved so that they can be used to inseminate females over an extended period of time (e.g. several days to one week). To inseminate many females with a single ejaculate the neat semen must be extended so that each inseminate dose contains less sperm than the entire ejaculate. Typically, the solution into which spermatozoa are diluted is referred to as an extender because it not only "extends" the number of sperm in the original ejaculate, but it "extends" their functional life. Extenders may be purchased from commercial sources or they can be prepared in the laboratory.

The Extender Must be Isotonic

You will recall from your basic biology class that when a cell is in an isotonic solution there is no net movement of water into or out of the cell. A hy- potonic solution is a solution in which the medium contains fewer osmotically active particles than the cell and water rushes into the cell and the cell membrane ruptures (cell lysis). In contrast, a hyper·tonic solution contains more osmotically active particles than does the inside of the cell and water moves out of the cell and it dehydrates. Providing the proper osmotic balance of the seminal extender is obligatmy for survival of spennatozoa.

An Extender Must Buffer and Protect

A buffer is a material that prevents marked changes in pH (hydrogen ion concentration). Extremes in pH, both acidic and alkaline, are damaging to all cells including spemmtozoa.

The cell membrane of a spem1atozoon is quite sensitive to sudden drops in temperature ("cold shock"). Care must be taken to prevent sudden declines in tem- perature so that the cell membrane and motile apparatus of the sperm do not become damaged. In neat semen, particular care must be taken to prevent damage to the spermatozoa. The design of the artificial vagina is important so that "cold shock" can be prevented (See Chapter I I). Slow, controlled cooling of sperm is important because it lowers the temperahtre gradually and minimizes stresses on the cell membrane. A low storage temperature reduces metabolism by about 50% for each 1 O"C decline. Spem1 are analogous to a battery. They have no option but to "run down." Unfortu- nately, recharging sperm cells after ejaculation is not possible.

Spermatogenesis 223

Where the goal is to extend the semen for a sustained period oftime (I week to years), a cryopro- tectant in the extender is required. Cryoprotectants are materials that protect the cells against cold damage that would occur between 0 and -50"C. These compounds protect spem1 membranes by minimizing ice crystal for- mation within the cell. In general, cyroprotectants can be classified as cell-permeating (glycerol, DMSO) and non-permeating (milk protein and egg yolk lipoprotein). Depending on species, one or a combination of types of cryoprotectants may be optimum. Common cryopro- tectants are glycerol and dimethyl sulfoxide (DMSO) with glycerol being the dominant ctyoprotectant for frozen sperm. Physiologic fluids are used frequently as extender ingredients. These include hen's egg yolk and cow's milk. These provide macromolecules that minimize cold damage and provide nutrients.

The rate of temperature decline and ultimate storage temperature are important depending on spe- cies. For example, a slow decline in temperature is important in the bull and the stall ion but is of much less importance to the dog and human. The influence of holding temperatures for unfrozen spetm also vaty among species. For example, bull and stallion semen can be stored effectively at 5"C while boar semen re- quires 18"C for best preservation. These differences are due, at least in part, to differences in lipid composition of the spenn membranes.

Spermatozoa have no anabolic capabi lity. In other words, spennatozoa are incapable of synthesizing materials for energy and repair. Therefore, the viability of sperm is totally dependent on the environment in which they are suspended. Nutrients need to be sup- plied in adequate quantities so that metabolism can be maintained for the appropriate duration of time. The major nutrients for spenn metabolism are fructose and glucose. Sperm are capable of converting glucose to fructose and metabolizing it to fuel their motility.

Ejaculated Semen is Not Sterile

Bacteria are present in the sheath and on the penis of the male and occasionally in the urethra and vesicular glands and therefore semen contains a variety of microorganisms. Seminal plasma and extender are ideal mediums for microbial growth and steps must be taken to mi nimize this growth. Antibiotics typically are added to the neat semen and extender to prevent micro- bial growth. Antibiotics such as penicillin, liquamycin, linco-spectin and streptomycin may be added in some combination to neat semen and to extenders.

Preservation of spermatozoa can be accom- plished using two methods. For relatively short term use, fresh liquid semen is used after the semen has been extended. In most species, liquid semen can be cooled

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222 Spermatogenesis

Immediately after collection, the following information is needed:

• ejaculate volume

• concentration of spermatozoa in the ejaculate (sperm/mL ejaculate)

• percentage of motile sperm

In Vitro Preservation is Obligatory for Successful AI

Evaluation of Semen is Needed Before Dilution

Total Sperm in Ejac. = Ejac. Vol. x Sperm/ml

Knowing the total number of sperm in the ejaculate enables the laboratory technician to determine how many insemination doses are potentially avai lable within each ejaculate.

Ejaculate volume = 6 ml

Sperm concentration = 1.0 X I 09 sperm/ml (I billion)

Total sperm in ejaculate = 6 ml x I .Ox I 09sperm/m i = 6x I 09 (6 billion)

Progressive motility = 70%

Total motile sperm= 6.0xl09 x.7 = 4.2x I09 motile sperm/ejaculate

Desired concentration = 15x I 06/dose ( I insemination)

Number of doses= 4.2x I 09/15x I 06 = 280 doses

Good seminal extenders must:

• be isotonic • be good buffers • minimize cold damage ("cold shock'') • provide appropriate nutrients • prevent microbial growth • maintain viability • be relatively low in cost

Seminal Extenders Extend Both Sperm Viability and Numbers

The Extender Must be Isotonic

An Extender Must Buffer and Protect

A buffer is a material that prevents marked changes in pH (hydrogen ion concentration). Extremes in pH, both acidic and alkaline, are damaging to all cells including spemmtozoa.

Spermatogenesis 223

Ejaculated Semen is Not Sterile

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224 Spermatogenesis

Table 10-3. Offspring Ratios of Spermatozoa Sorted for the X and Y Chromosome

Species Sorted for Y Chromosome Sorted for X Chromosome %Male %Female %Male %Female

Cattle 81 Rabbit 81 Swine 75

and stored at near freezing temperature ( 5°C) for several days to about one week. In swine, 17 -18"C is optimum. When widespread distribution and long-term usage is a requirement, frozen semen is the prefened method of preservation. When frozen semen is used, careful attention to freezing and thawing techniques must be practiced. Freezing and thawing compromises sper- matozoal viability in all species. However, the degree to which viability and fertility are affected depends on the individual male and species.

Sex of the Conceptus is Determined by the Sperm Because Each Spermatozoon Contains

Either an X or a Y Chromosome

As you already know, each secondary sperma- tocyte produces two haploid daughter spennatids. Each spermatid contains either an X or a Y chromosome. Sperm containing the X chromosome that fertilize an oocyte will generate a female. Spenn containing the Y chromosome will generate a male (See Figure 10-15).

The desire to separate the X and Y bearing spenn is driven by the fact that one sex has signifi- cantly more economic value than the other in certain species. For example, in the dairy industry, bull calves are of little value since about 80% of all cows in the U.S. (and higher on a worldwide basis) are artificially inseminated. Thus, relatively few bulls are required to inseminate the cows in the national dairy herd. The cow is the primary income generator for a dairy business. It would be advantageous to have a high percentage of female offspring since lactation is limited to the female. In other food producing animals, it might be more desirable to produce higher percentage males since these animals grow faster and have more desired meat characteristics.

The X and Y chromosome contain differ- ent quantities of DNA. For example, an X bearing spenn contains 2.8-4.2% more DNA (depending on the species) than does a Y bearing sperm. Based on this difference, it is possible to separate the X and Y bearing spenn into two subpopulations. The separation

19 11 89 19 6 94 25 10 90

procedure requires the uptake of a DNA stain or dye (called a flourochrome) into both living and dead sper- matozoa. Those sperm that contain the X chromosome "take-up" more DNA dye than do sperm containing the Y chromosome. Vital dyes used to stain sperm produce emissions oflight at a specific wavelength when excited or activated by light at a specific wavelength.

The technology utilized for separation of X and Y bearing spennatozoa is referred to as flow cytometry (sometimes called "cell sorting"). Figure I 0-15 high- lights the major steps for separation of the X and Y bearing spem1atozoa using flow cytometry.

Experimental evidence clearly shows the success of this technology for separating the X and Y bearing spermatozoa from common mammals and most experiments have yielded 80-90% successful separa- tion for either males or females in cattle, swine and rabbits (See Table 1 0-3). Several factors have limited the efficiency of this technology, but the application is now widespread in dairy cattle and is feasible in many other species.

Regardless of the problems associated with separating the X andY bearing spermatozoa, the tech- nology is now available through artificial insemination organizations. Thus, it is reasonable to expect that separation of X andY bearing spermatozoa eventually will be commonplace. Manipulation of the sex ratio under controlled conditions could greatly impact the efficiency offood animal production.

Spermatogenesis 225

Figure 10-15. Major Steps for Separation of X andY Bearing Spermatozoa by Flow Cytometry

Step 1 X and Y bearing spermatozoa are produced by the testis and ejaculated by the male.

Step 2 Ejaculated spermatozoa are treated with a fl uorescent DNA dye. X bearing sperm absorb more dye than Y beanng. sperm. They therefore emit more intense light when exc1ted by a laser. Sperm also are treated with a dye that greatly suppresses the signal from dead sperm. Dead sperm are therefore identified and rejected.

Step 3 Once enter the flow cytometer chamber, they

smgle-file through a small nozzle. At a region just out- Side the nozzle, a laser beam excites the fluorescent dye in

sper£!1. Eac.h sperm emits light at a wavelength and that IS directly related to quantity of DNA. X-

live sperm produce more intensity. A light sensing dev1ce 1s coupled to a computer that determines the intensity of light emission by each sperm and the order of passage of each sperm through a column below the nozzle. When the sperm pass by charged plates, they are assigned either a positive or negative charge depending on their DNA content

or Y chromosome). When the microdroplet containing a s1ngle sperm passes through an electromagnetic field the computer applies an appropriate charge and directs the

(and to one side or the other. Dead sperm are discarded 1nto the center tube. Thus, at the conclusion of the separation process there are three vessels that contain sperm. One contains a high proportion of X one contains a high proportion of Y chromosome bearing and one contains dead sperm.

Ve tB oo ks .ir

224 Spermatogenesis

Table 10-3. Offspring Ratios of Spermatozoa Sorted for the X and Y Chromosome

Species Sorted for Y Chromosome Sorted for X Chromosome %Male %Female %Male %Female

Cattle 81 Rabbit 81 Swine 75

Sex of the Conceptus is Determined by the Sperm Because Each Spermatozoon Contains

Either an X or a Y Chromosome

19 11 89 19 6 94 25 10 90

Spermatogenesis 225

Figure 10-15. Major Steps for Separation of X andY Bearing Spermatozoa by Flow Cytometry

Step 1 X and Y bearing spermatozoa are produced by the testis and ejaculated by the male.

Step 3 Once enter the flow cytometer chamber, they

smgle-file through a small nozzle. At a region just out- Side the nozzle, a laser beam excites the fluorescent dye in

sper£!1. Eac.h sperm emits light at a wavelength and that IS directly related to quantity of DNA. X-

or Y chromosome). When the microdroplet containing a s1ngle sperm passes through an electromagnetic field the computer applies an appropriate charge and directs the

Ve tB oo ks .ir

226 Spermatogenesis

Further PHENOMENA for Fertility

Spermatozoa of the American opossum are ejaculated in doublets. They are formed in the seminiferous epithelium as single cells with an acrosome. During epitlidymal tran- sit the acrosome of two spermatozoa attach to each other, so that a pair of spermatozoa exists. These doublets apparently have more progressive motility than do single cells. When motility ceases they apparently separate.

In Greek Mythology, when Priapus was in the womb of his mother Aphrodite, Hem put a spell on him to make him ugly. When he was bom, he was of small stature am/ very ugly but possessed an extremely large penis that was always erect. The name Priapus gave rise to the medical term "priapism", which is defined as a persistent (sometimes painful) erection of the penis, associated with some form of pathology (blood clot in cavernous tissue) and not sexual e.'Ccita- tion. Priapus became known as the Greek God of Fertility in most species, including plants, animals and humans. As the source of fertility, statues of Priapus were kept itt gardens to ensure fertile crops am/ to scare away thieves. He has also been thought to be a cm·e for impotence.

In some regions of the world, testes are prized as gourmet treats. In Japan, tes- ticles of dolphins m·e highly valued hors d'oeuvres. In Spain, bull testicles are served at social eJ,ents surrounding the occasion of a bullfight. Bull testicles are also consumed by hungry American cowboys at castration time. In all cases, they are cooked.

The bulls at a leading AI organization produce a lot of sperm. The annual semen production from the bulls collected is as follows:

• 42-43,000 ejaculations per year • 205 trillion spermatozoa per year

• 454 lbs. (206kg) neat semen per year

• 10,282,759 0.5-ml straws per year • 12,196lbs. (6.1 tons) of extended

semen pe1· year

It is rumored that during the early stages of Christianity, the church had succeeded in getting the pagans to give up worship of all the old gods e.'Ccept P1·iapus (the Greek God of Fertility with a huge penis). No matter what the threats or enticements were, the loyal worshipers of Priapus would not give up reverence for their favorite god. The expression of this unwavering reverence included the baking of bread in the shape of a penis 011 eveiJ' available celebrat01y occasion, including church holidays. Un- able to dissuade the people from this rather un-Christian practice, the wise church fa- thers sanctified the loaves, providing each had three crosses carved into its top. This was the reported beginning of hot crossed buns.

The ancient Greeks thought that sperma- tozoa from the left testicle produced girls and spermatozoa from the right testicle pro- duced boys. This myth apparently stood the "test of time" because as late as the 1700s, French noblemen would have their left testicle removed in an attempt to sire boys only. The author proposes that the modem day declaration by males, "I would give my left testicle for a ---", is a sexist comment that devalues the left testicle because it was once thought to produce females only. Have you ever heard a male say he was willing to give-up his right testicle for something?

Lazzaro Spal/anzani was a mathematician and He was also a priest who conducted experiments with sperm and eggs. His religious beliefs prevented him from collecting and working with human sperm. He wondered, tltouglt, if eve1y 1m- man sperm in an ejaculate hat! a soul and, if so, what happened to the millions of souls in wasted semen. If eve1y sperm had its own soul, then masturbation and contraception were serious sins . . In Spallanzani's era

(1700s) many biologists believed in "re- mote fertilization," in which the egg could be stimulated to develop without contact with semen. They thought if an egg were exposed to invisible "spermatic vapor" it would develop into an emb1yo. Since "spermatic vapor," like a ghost, could not be seen there was some wony that this ghostly vapm; once released from an ejaculate, might waft-up the legs of some unsuspecting female, causing an unwanted pregnancy. No one knows how many 1mman·ied women of Spallanzani's era may have credited their pregnancies to "spermatic vapor. " Spa/lanzmzi beliel'ed in these sperm ghosts . but wanted to test his belief. He attached fi'eshly laid toad eggs to a watch glass and inverted it over another watch glass con- taining toad seminal fluid. He thus had an enclosed system in which the eggs and semina/fluid were separated, and where the invisible "spermatic vapor" could migrate to stimulate the eggs. Not/zing happened. But when the eggs were mixed directly into semina/fluid, the physical contact produced tadpoles. How ditl Spallanzani obtain frog semen? He dressed male frogs in tiny taf- feta trousers and placed them with a female frog (without clothes). He watelproofed the pants with a light coating of candle wax. Aroused, the males mounted the females and ejaculated in their pants. Spallanzani then collected the semen. These experi- ments might have been the forerunners to in vitro fertilization because tadpoles developed. Spallanzani was one oftlte first scientists to achieve artificial collection of semen in a laboratory under controlled conditions.

Spermatogenesis 221

Kev References

Amann, R.P. 1999. "Cryopreservation of sperm" in Encvc/opedia o(Reproduction Vol. 1 p 773-783. Knobil, E. and J.D. Neill (eds). Academic Press, San Diego. ISBN 0- 12-227021 -5.

Barth, A.D. and R.J. Oko. 1989. Abnormal MomholofJY o{Bovine Spermatozoa. Iowa State University Press, Ames. ISBN 0-8138-01 12-5.

Dadoune, J.P. and A. Demoulin. 1993. "Stmcture and fu nctions of the testis" in Reproduction in Mammals and Man . C. Thibault, M.C. Levasseur and R. H. F. Hunter, eds. Ellipses, Paris. ISBN 2-7298-9354-7.

Ericsson, R.J. and S.A. E1i csson. 1999. "Sex ratios" in Encvc/opedia o(Reproduction. Vol. 4 p 431 -436. Knobil, E. and J.D. Nei ll (eds.) Academic Press, San Diego ISBN 0- 12-227024-X.

Hess, R.A. 1999. "Spermatogenesis overview" in En- cvc/opedia o(Reproduction. Vol. 4 p 539-545. Knobil, E. and J.D. Nei ll (eds). Academic Press, San Diego. ISBN 0- 12-227024-X.

Johnson, L. I 991. "Spermatogenes is" in Reproduction in Domestic Animals 4th Edition, P.T. Cupps, ed. Aca- demic Press, Inc. San Diego. ISBN 0-12-1 9657 5-9.

Johnson, L. T.A. McGowen, G.E. Keillor. 1999. "The Testis, overview" in Encvc/opedia o{Reproduction, Vol. 4 p 769-783. Knobil and NeiiJ , eds. Academic Press, San Diego. ISBN 0-12-227024-X.

Lamming, G.E. ed. 1990. Marshal/'s PhvsiolofJY of Reproduction. Fourth Edition Vol. 2: Reproduction in the Male. Churchill Livingstone, New York. ISBN 0-443-0 1968-1 .

Russel, L. D. and M. D. Griswold, eds. 1993. The Serto/i Ce/1. Cache River Press, Clearwater. ISBN 0-9627422-0-1-X.