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8.2: Meiosis and Gametogenesis (Instructor Materials Preparation) - Biology


Lab Materials

This is the prep for one section of 24 students.

Part 1: Meiosis Bead Simulations

Students will do this part in table teams.

MaterialsQuantityNotes
Chromosome bead sets

Part 2: Independent Assortment

Students will do this part in table teams.

MaterialsQuantityNotes
Chromosome bead sets

Part 3: Mammalian Gametogenesis

Students will do this part in table teams.

MaterialsQuantityNotes
Slides of ovaries1 boxHN 1/21
Slides of testes1 boxHM 1/2

EXTRA SUPPLIES: Models

Students will do this part in table teams.

MaterialsQuantityNotes
Mitosis models
DNA models2

AP Biology : Understanding Gametogenesis

Which answer best explains why each primary spermatocyte cell (2n) results in the eventual production of four spermatids (n), while each oogonium cell (2n) results in the eventual production of only one active egg cell (n)?

During spermatogenesis, the cellular material is divided evenly during meiosis I and II, resulting in four equally sized spermatids. During oogenesis, both meiosis I and meiosis II involve the uneven distribution of cytoplasm, which results in only one active egg cell and two or three polar bodies.

During spermatogenesis, the cellular material is divided evenly during meiosis I and II, resulting in four equally sized spermatids. During oogenesis, only one of the two daughter cells that results from meiosis I passes through meiosis II the other daughter cell remains dormant. Of the daughter cells that result from meiosis II, one becomes an active egg cell while the other disintegrates.

During spermatogenesis, the cellular material is divided evenly during meiosis I and II, resulting in four equally sized spermatids. During oogenesis, the process of meiosis results in four potential active egg cells of equal size and functionality arbitrarily, three of these cells are degraded, leaving only active egg cell.

None of the other answers are correct

During spermatogenesis, the cellular material is divided evenly during meiosis I and II, resulting in four equally sized spermatids. During oogenesis, both meiosis I and meiosis II involve the uneven distribution of cytoplasm, which results in only one active egg cell and two or three polar bodies.

Spermatogenesis is a continuous process starting at puberty. Sperm production takes place in the seminiferous tubules. There, each spermatogonium cell (2n) divides equally via mitosis to produce two identical primary spermatocytes (2n). Each primary spermatocyte undergoes meiosis I to produce two equally sized secondary spermatocytes (n). Each secondary spermatocyte then undergoes meiosis II to produce a total of four spermatids (n). The spermatids differentiate before moving to the epididymis.

Oogenesis, on the other hand, begins prior to birth and is a stop-start process. While still within the embryo, each oogonium (2n) produces two identical primary oocytes (2n) via mitosis the primary oocytes remain dormant in small ovarian follicles until puberty. After the onset of puberty, hormones periodically stimulate the follicles to complete meiosis I and produce secondary oocytes (n). It is important to note, however, that meiosis I produces only one secondary oocyte—the other "daughter cell" is actually the first polar body, which is almost always rapidly degraded (though sometimes this polar body also undergoes meiosis II and produces a third polar body). This occurs because the primary oocyte divides unevenly, leaving the secondary oocyte with virtually all of the cytoplasm during cytokinesis. Secondary oocytes are released during ovulation, but they do not complete meiosis II until fertilization, when they are penetrated by a sperm. Meiosis II results in another polar body, again due to uneven division of cytoplasm during cytokinesis.

Example Question #1 : Understanding Gametogenesis

Which term refers to the formation of egg cells that begins in the developing ovaries of a female fetus?

The mature human ovum is formed through the developmental process known as oogenesis, which takes place during the first three months of fetal development. Ovarian follicles each contain one oogonium, which becomes a primary oocyte, then a secondary oocyte. No further division occurs until puberty.

Mitosis is a type of nuclear division in which one copy of each chromosome moves into each of two daughter nuclei to create identical daughter cells. Meiosis is also a type of cell division, in which a diploid cell divides twice to produce four haploid cells. Ovulation is the release of the secondary oocyte from the ovary, and fertilization is the union of a male sperm and female egg to produce a zygote.

Example Question #1 : Understanding Gametogenesis

Spermatogenesis is the production of sperm by the process of __________ , followed by differentiation.

During spermatogenesis, spermatogonia become primary spermatocytes, then seconday spermatocytes. These divide to form two spermatids, which transform into functional spermatozoa. Through the process of meiosis, the chromosome number is reduced from the diploid number (46) to the haploid number (23).

Mitosis refers to the replication of somatic cells, creating identical daughter cells from a diploid parent. Migration occurs as the spermatogonia, spermatocytes, and spermatids move from the outermost edge of the seminiferous tube to the central cavity of the tuble. Proliferation is the repeated reproduction of new parts. Gametogenesis is defined as the development of gametes.

Example Question #51 : Reproductive System

Mitosis and meiosis differ in that mitosis produces __________ cells with __________ genetic material, whereas meiosis produces __________ cells with __________ genetic material.

4 . . . a unique mixture of . . . 2 . . . identical

2 . . . identical . . . 4 . . . identical

2 . . . identical . . . 4 . . . a unique mixture of

2 . . . a unique mixture of . . . 2 . . . identical

2 . . . identical . . . 4 . . . a unique mixture of

During mitosis cells divide a single time and retain the exact same genetic material, producing two identical copies of the parent cell. During meiosis, cells divide twice and cross over during anaphase I. This produces a unique combination of chromosomes (recombinants) not seen in the parent cell.

Example Question #51 : Reproductive System

In humans the gametes are produced in __________ .

Gametes are the haploid sex cells that are produced in the gonads (ovaries in females and testes in males). These are the primary sex organs.

Example Question #58 : Reproductive System

A human cell from the ovary has 22 chromosomes and an X chromosome. It is __________ .

A cell with 22 chromosomes and 1 sex chromosome is clearly haploid (n=23) and so it must be a sex cell and not a somatic cell (2n=46). Since both male and female gametes may contain an X chromosome, that information is not enough to tell us whether this cell comes from a male of female. However, since we are told the cell came from the ovary, we know it is the female gamete, an ovum. A muscle cell is a type of somatic cell, all of which are diploid.

Example Question #59 : Reproductive System

Which of the following is not a true characteristic of gametocytes?

Male gametocytes are called spermatocytes

Gametocytes can divide by mitosis into other gametocytes

They are eukaryotic somatic cells

Female gametocytes are called oocytes

They are eukaryotic somatic cells

Gametocytes are eukaryotic germ line cells. They can undergo mitosis to form more gametocytes or undergo meiosis to form gametids. Male gametocytes are called spermatocytes and female gametocytes are called oocytes.

Example Question #60 : Reproductive System

Which of the following best describes the difference between a primary and secondary spermatocyte?

The secondary spermatocyte is haploid and the primary spermatocyte is diploid

The secondary spermatocyte forms during mitosis and the primary spermatocyte forms during meiosis II

The secondary spermatocyte forms during meiosis II and the primary spermatocyte forms during mitosis

The secondary spermatocyte is diploid and the primary spermatocyte is haploid

The secondary spermatocyte is haploid and the primary spermatocyte is diploid

Spermatocytes are male gametocytes located in the seminiferous tubules of the testes. Primary spermatocytes are diploid and form when spermatogonia—immature germ cells—enter into mitosis. Primary spermatocytes can then enter meiosis and produce haploid secondary spermatocytes after meiosis I.

Example Question #61 : Reproductive System

Which of the following is not a true characteristic of spermatogonia?

They develop into primary spermatocytes through mitosis

They are undifferentiated

Spermatogonia are diploid undifferentiated male germ cells located in the seminiferous tubules of the testes. Spermatogonia are important in the process of spermatogenesis they turn into primary spermatocytes via growth and maturation.

Example Question #62 : Reproductive System

Which of the following terms is best defined as a mature, motile, and haploid sperm cell produced during spermatogenesis?

Mature sperm cells that are haploid, motile, and produced during spermatogenesis are called spermatozoa. Spermatids are haploid however, they are immature. Spermatids will turn into spermatozoa once they mature.

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Male Reproductive Anatomy

In the male reproductive system, the scrotum houses the testicles or testes (singular: testis), including providing passage for blood vessels, nerves, and muscles related to testicular function. The testes are a pair of male reproductive organs that produce sperm and some reproductive hormones. Each testis is approximately 2.5 by 3.8 cm (1.5 by 1 in) in size and divided into wedge-shaped lobules by connective tissue called septa. Coiled in each wedge are seminiferous tubules that produce sperm.

Sperm are immobile at body temperature therefore, the scrotum and penis are external to the body, as illustrated in Figure 24.8 so that a proper temperature is maintained for motility. In land mammals, the pair of testes must be suspended outside the body at about 2 ° C lower than body temperature to produce viable sperm. Infertility can occur in land mammals when the testes do not descend through the abdominal cavity during fetal development.

Which of the following statements about the male reproductive system is false?

  1. The vas deferens carries sperm from the testes to the penis.
  2. Sperm mature in seminiferous tubules in the testes.
  3. Both the prostate and the bulbourethral glands produce components of the semen.
  4. The prostate gland is located in the testes.

Sperm mature in seminiferous tubules that are coiled inside the testes, as illustrated in Figure 24.8. The walls of the seminiferous tubules are made up of the developing sperm cells, with the least developed sperm at the periphery of the tubule and the fully developed sperm in the lumen. The sperm cells are mixed with “nursemaid” cells called Sertoli cells which protect the germ cells and promote their development. Other cells mixed in the wall of the tubules are the interstitial cells of Leydig. These cells produce high levels of testosterone once the male reaches adolescence.

When the sperm have developed flagella and are nearly mature, they leave the testicles and enter the epididymis, shown in Figure 24.8. This structure resembles a comma and lies along the top and posterior portion of the testes it is the site of sperm maturation. The sperm leave the epididymis and enter the vas deferens (or ductus deferens), which carries the sperm, behind the bladder, and forms the ejaculatory duct with the duct from the seminal vesicles. During a vasectomy, a section of the vas deferens is removed, preventing sperm from being passed out of the body during ejaculation and preventing fertilization.

Semen is a mixture of sperm and spermatic duct secretions (about 10 percent of the total) and fluids from accessory glands that contribute most of the semen’s volume. Sperm are haploid cells, consisting of a flagellum as a tail, a neck that contains the cell’s energy-producing mitochondria, and a head that contains the genetic material. Figure 24.9 shows a micrograph of human sperm as well as a diagram of the parts of the sperm. An acrosome is found at the top of the head of the sperm. This structure contains lysosomal enzymes that can digest the protective coverings that surround the egg to help the sperm penetrate and fertilize the egg. An ejaculate will contain from two to five milliliters of fluid with from 50–120 million sperm per milliliter.

Figure 24.9. Human sperm, visualized using scanning electron microscopy, have a flagellum, neck, and head. (credit b: modification of work by Mariana Ruiz Villareal scale-bar data from Matt Russell)

The bulk of the semen comes from the accessory glands associated with the male reproductive system. These are the seminal vesicles, the prostate gland, and the bulbourethral gland, all of which are illustrated in Figure 24.8. The seminal vesicles are a pair of glands that lie along the posterior border of the urinary bladder. The glands make a solution that is thick, yellowish, and alkaline. As sperm are only motile in an alkaline environment, a basic pH is important to reverse the acidity of the vaginal environment. The solution also contains mucus, fructose (a sperm mitochondrial nutrient), a coagulating enzyme, ascorbic acid, and local-acting hormones called prostaglandins. The seminal vesicle glands account for 60 percent of the bulk of semen.

The penis, illustrated in Figure 24.8, is an organ that drains urine from the renal bladder and functions as a copulatory organ during intercourse. The penis contains three tubes of erectile tissue running through the length of the organ. These consist of a pair of tubes on the dorsal side, called the corpus cavernosum, and a single tube of tissue on the ventral side, called the corpus spongiosum. This tissue will become engorged with blood, becoming erect and hard, in preparation for intercourse. The organ is inserted into the vagina culminating with an ejaculation. During intercourse, the smooth muscle sphincters at the opening to the renal bladder close and prevent urine from entering the penis. An orgasm is a two-stage process: first, glands and accessory organs connected to the testes contract, then semen (containing sperm) is expelled through the urethra during ejaculation. After intercourse, the blood drains from the erectile tissue and the penis becomes flaccid.

The walnut-shaped prostate gland surrounds the urethra, the connection to the urinary bladder. It has a series of short ducts that directly connect to the urethra. The gland is a mixture of smooth muscle and glandular tissue. The muscle provides much of the force needed for ejaculation to occur. The glandular tissue makes a thin, milky fluid that contains citrate (a nutrient), enzymes, and prostate specific antigen (PSA). PSA is a proteolytic enzyme that helps to liquefy the ejaculate several minutes after release from the male. Prostate gland secretions account for about 30 percent of the bulk of semen.

The bulbourethral gland, or Cowper’s gland, releases its secretion prior to the release of the bulk of the semen. It neutralizes any acid residue in the urethra left over from urine. This usually accounts for a couple of drops of fluid in the total ejaculate and may contain a few sperm. Withdrawal of the penis from the vagina before ejaculation to prevent pregnancy may not work if sperm are present in the bulbourethral gland secretions. The location and functions of the male reproductive organs are summarized in Table 24.1.

Table 24.1.
Male Reproductive Anatomy
Organ Location Function
Scrotum External Carry and support testes
Penis External Deliver urine, copulating organ
Testes Internal Produce sperm and male hormones
Seminal Vesicles Internal Contribute to semen production
Prostate Gland Internal Contribute to semen production
Bulbourethral Glands Internal Clean urethra at ejaculation

Results

Generation of the Prdm9-deficient rats

Although some mammalian genomes carry multiple PRDM9-encoding genes, the rat genome was reported to contain only a single-copy Prdm9 gene [24]. To decipher the necessity of Prdm9 for rat fertility, we targeted this gene in the SHR/OlaIpcv strain (SHR see Methods) in one of the exons encoding the PR/SET domain. This domain catalyzes both H3K4 [12] and H3K36 trimethylation in human and mouse [25, 26]. The mRNAs of a programmed heterodimeric nuclease were injected into 234 fertilized ova (Table 1). Four animals carrying deletions of two, eight, 39, and 516 base pairs in size (bp) were generated the two largest ones also affected one exon-intron boundary. To assess the effects of these deletions on Prdm9 mRNA, we performed reverse-transcribed (RT) PCR with primers from the exon sequences surrounding the deletions using testicular RNA. Sequencing of these RT-PCR products from testicular RNA revealed that the three shortest deletions resulted in the shifts of PRDM9 open reading frames, producing putative PR/SET domain truncations and exclusion of the PRDM9 zinc-finger DNA-binding domain (Fig. 1). The mutant with the 516-bp deletion produced two new transcripts, one with a frame shift and one with an internal deletion of 20 codons of the PR/SET catalytic domain that retained the PRDM9 zinc-finger-encoding exon in frame (Fig. 1, Additional file 1: Fig. S1). All deletions removed one or more amino acid residues important for the methyltransferase activity of PRDM9 ([27] Additional file 1: Fig. S1). Rats genotyped homozygous for these mutations using genomic DNA were also homozygous when probed by RT-PCR using testicular mRNA, supporting whole genome sequence assembly data that suggested that Prdm9 is present in a single copy in the rat [24, 28]. Semi-quantitative RT-PCR of testicular RNA revealed that males homozygous for the Prdm9 deletions of 2-, 8-, and 39-bp display similar mRNA levels as littermate controls and confirmed expression of the two new mRNA isoforms from the mutant with the 516-bp deletion (Additional file 1: Fig. S2).

Four Prdm9 deletions generated in SHR rats lead to mRNAs with open reading frame truncations and one also to alternative mRNA with deletion of 20 codons. a mRNAs arising from rats with genomic deletions of 2-, 8-, 39-, and 516-bp (abbreviated KO2, KO8, KO39, and KO516, respectively the latter two also involve a part of Intron 8) blue dashes, gaps to optimize the alignment red dashes, exonic deletions -U, -L, two new mRNAs from the animal with the KO516 deletion. b Polypeptide products predicted from mRNAs in a and their detection. -20 a.a., deletion of 20 amino acid residues. The C-terminal boxes indicate zinc-fingers and the three letters their DNA-binding amino acids. See Additional file 1: Fig. S1 for details of the translations and amino acids essential for methyltransferase activity and Additional file 2: Table S1 for genomic and cDNA sequences. Right, PRDM9 was detected at the expected sizes of 97 kDa (mouse Dom2 allele) and 96 kDa (rat SHR allele). Anti-SYCP3 antibody was utilized as a loading control. PRDM9 was present in the SHR-Prdm9 KO516/KO516 mutants at the expected size of the KO516-L isoform (91 kDa), but its expression was lower than that of the wild-type in SHR the KO516-U isoform (predicted 40 kDa) was undetectable. KO39 mutant product was not found (expected 44 kDa) in the SHR-Prdm9 KO39/KO39 testes, thus no C-terminally truncated form of PRDM9 was seen in either mutant. Only a third of the amount of protein nuclear extract was loaded in mouse (10 μg) compared to rat (30 μg) lanes

To evaluate PRDM9 protein expression in wild-type and mutant testes, we performed Western blotting (Fig. 1b). PRDM9 was detected SHR testes at 18 days post partum (dpp), where most germ cells are pre/leptotene and zygotene spermatocytes (see below for details on 18-dpp testes). Neither PRDM9 protein nor its truncated form was found in 18-dpp SHR-Prdm9 KO39/KO39 rat testes, suggesting that its frame-shifted mRNA is not translated or quickly degraded. PRDM9 was present in 18-dpp SHR-Prdm9 KO516/KO516 rats at the size of 91 kDa predicted from the mRNA with the internal deletion of 20 codons of the catalytic PR/SET domain, but not at the size of 40 kDa expected from the alternatively spliced mRNA with the frame shift. However, the expression of the 91 kDa product was lower than the expression of the wild-type PRDM9.

Inactivation of rat Prdm9 is compatible with fertility in both sexes

We next tested the fertility of the rats carrying the Prdm9 deletions. Twenty-one of 22 (95%) of Prdm9-deficient and all 26 tested heterozygous males sired offspring. Heterozygous Prdm9 KO/wt males that produced at least two litters (n = 26) sired 6.8 ± 1.6 (mean ± standard deviation) of offspring per female per month, 32% more compared to the Prdm9-deficient males with at least two litters (n = 15 4.6 ± 2.1 P = 0.0009 Welsch’s t test Fig. 2f). The litter size itself also decreased in Prdm9-deficient males (Fig. 2e). Adult (71 to 98 days old) Prdm9-deficient males had relative testicular weight and sperm count reduced by about 40% compared to both heterozygote and wild-type rats (P < 0.0001 each linear regression model (LRM) Fig. 2a–c, Additional file 1: Fig. S3).

Decreased fertility parameters of SHR males carrying Prdm9 deletions depend on age. Each dot depicts a single animal. a–c Boxplots of sperm count (a), testicular weight (b), and relative testicular weight (c, mg testicular weight per gram of body weight). d, Representative images of hematoxylin-eosin (HE) stained sections from rat testicles note the presence of mature spermatids (arrows) in all three males. e Litter size of male parents of two indicated Prdm9 genotypes for paternal age at birth. The statistical significance of the differences between genotypes was tested using LRM in the age groups of up to 150 and above 150 dpp. f Number of pups per month per female (OMU) for mating ranges (days) in SHR males of two indicated Prdm9 genotypes. The data underlying all published plots are given in Additional file 3

Prdm9-deficient adult females (69 to 373 days old) had decreased ovary weight and relative ovary weight compared to controls (P < 0.0001, LRM accounting for age Fig. 3a, b, Additional file 1: Fig. S3). Of 25 Prdm9-deficient females crossed for at least 2 months, 11 (44%, carrying all four deletion types) produced offspring, while all 27 tested Prdm9 KO/wt females yielded pups. Moreover, the litter size of the Prdm9-deficient (Prdm9 KO/KO ) female parents (5.4 ± 1.7) was smaller than that of Prdm9 KO/wt (8.3 ± 1.6 P = 0.0003 Welsch’s t test Fig. 3d). The pups generated by both the male and female Prdm9 KO/KO rats were fertile and appeared normal. No offspring has been recovered when both mating partners were Prdm9-deficient (7 females, 2-month crosses). In summary, in contrast to B6 and PWD mice, both male and female SHR rats lacking PRDM9 function display only a partial reduction of fertility.

Decreased fertility and premature ovarian failure in SHR females carrying Prdm9 deletions. a, b Boxplots of ovary weight (a, in mg) and of relative ovary weight (b, mg ovary weight per gram of body weight). c Examples of HE stained sections from three adult rat ovaries at two magnifications. Note that all females carry germ cell follicles (arrows). d Litter size of female parents of two indicated Prdm9 genotypes with maternal age at birth. The difference between genotypes was tested using the LRM in the age group of up to 150 dpp. e Number of pups per month per female (OMU) and mating ranges (days) for two indicated Prdm9 genotypes. The abscissa at OMU = 0 contains data for 14 Prdm9-deficient females, ten of which were mated after 150 dpp (for additional mean of 26 days). f Follicle quantification from ovarian sections of the indicated Prdm9 genotypes at three points of postnatal development. Differences between three Prdm9 genotypes and between animals of three ages within each genotype were analyzed using LRM with subsequent Tukey test and Bonferroni adjustment. g Representative images of HE-stained ovarian sections from 21-dpp females

Inactivation of rat Prdm9 induces four partial arrests of spermatogenesis

To uncover the reasons for male fertility reduction, we performed immuno-, histo-, and cyto- chemistry of the germline. Within an adult rat testis, subsequent waves of spermatogenesis begin once every

12 days (8–9 in the mouse) [29] the entire rat spermatogenesis takes

52 days. Premeiotic spermatogonia line the periphery of the seminiferous tubule, divide mitotically, and mature. The resulting primary spermatocytes proceed through meiotic prophase and two specialized divisions to yield spermatids, which differentiate into sperm until being released into the tubular lumen. Because a new spermatogenesis cycle begins before the previous cycles have ended, each tubule carries various germ cell types. As both the time between cycles and the duration of individual developmental stages are fixed, each rat tubule section can be classified into 14 types (12 in the mouse), referred to as stages I to XIV.

To identify the specific stages of the seminiferous tubules in the mutant testes affected by Prdm9 inactivation, we categorized and quantified cells in all 14 stages of rat tubules (over 60,000 cells) using sections from adult testes stained with periodic acid, Schiff, and hematoxylin (PAS-H, Fig. 4a and Fig. 5, statistics in Additional file 2: Table S2). Overall, all 14 tubule stages were readily identifiable and the counts of spermatogonia were similar in the mutant and control rat testes. However, the mutant tubules of all stages were more sparsely populated compared to controls (all 14 adjusted P values below 0.036 LRM), mostly due to lower counts of round and elongated spermatids (all 22 P < 0.037). These decreased numbers of spermatids could occur either solely due to partial meiotic arrest(s) or postmeiotic arrest(s) or both. To resolve these three possibilities, we compared abnormal cell counts. We found elevated counts of abnormal (halo [30], joint and degraded [31]) round spermatids in mutant stages IV (summary P = 0.027) and V (P = 0.028) compared to wild-type controls, which supports the contribution of the postmeiotic arrests (Fig. 6a). Moreover, we detected an increased number of defective mid-pachytene spermatocytes in stage IV (P = 0.022) and of degenerated metaphase spermatocytes in stage XIV (corresponding to mouse stage XII P = 0.015) of Prdm9-deficient testes, indicative of two types of meiotic arrests. In agreement with this conclusion, the mutant stages V to XII contained less pachytene spermatocytes (all eight P values < 0.024) and mutant stage XIV both less late primary spermatocytes (P < 0.0001) and less metaphase II spermatocytes (P = 0.020) compared to the controls. In addition, stages VIII, X, and XI of mutant tubules contained elevated numbers of leptotene spermatocytes (all three P < 0.035), which may indicate prophase I delay. Altogether, the histopathology results suggested that the ablation of the rat Prdm9 function caused partial arrests of meiosis at stages IV and XIV, a delayed meiotic prophase I, and incomplete spermiogenesis failures in stages IV and V.

Prdm9-deficiency causes spermatogenic arrests in tubular stages IV and XIV of SHR rat. a Testicular histology using PAS-H staining. Illustrative examples of all 14 rat tubule stages. Degenerated cells in seminiferous tubules from adult (71 dpp) mutants stain intensively with PAS-H (blue-purple) at stages IV and XIV (red arrows). P, pachytene spermatocytes RSP, round spermatids ESP, elongated spermatids S, Sertoli cells Pl, preleptotene L, leptotene D, diplotene Z-P, zygotene-pachytene spermatocytes MI, metaphase I spermatocytes. Quantification of cells in all stages is presented in Fig. 5. b Increased apoptosis in mutant rat stage IV and XIV tubules confirmed by labeling apoptosis (TUNEL), acrosome (PNA), and DNA (Hoechst 33342)

Prdm9-deficiency changes the quantities of spermatogenic cells in all 14 tubular stages of SHR rat. Cell counts of spermatogenic cell types in seminiferous tubules of stages I to XIV are given including the mean cell number ± standard deviation per tubule in each stage calculated from 3 to 5 tubules for each stage and animal (4 mutants and 3 controls) from sections stained with PAS-H. SG A, type A spermatogonia SG In, intermediated spermatogonia SG B, type B spermatogonia see legend of Fig. 4 for abbreviations of primary spermatocytes and spermatids MII, metaphase II spermatocytes sSC, secondary spermatocytes dM, degenerated MI/II and sSC dP, degenerated pachytene dSP, degenerated, halo, and joint spermatids

Rat PRDM9 affects postmeiotic development of round spermatids. a Examples of normal and defective spermatids from wild-type and Prdm9-deficient SHR males on PAS-H stained testicular sections. The means counted in mutant versus control in all 14 stages are in Fig. 5. b, c Increased apoptosis during postmeiotic development of Prdm9-deficient SHR testes. b Mean counts of apoptotic round spermatids per apoptotic tubule were scored from randomly chosen apoptotic tubules (18 to 32 per animal, total 221 tubules from 9 animals), plotted, and evaluated using LRM. The average counts of mutant and control samples were 1.1 ± 0.4 and 0.4 ± 0.2, respectively. c Round spermatids (RSP) were distinguished on neighboring TUNEL-DAPI and anti-PIWIL1-Hematoxylin-stained sections from spermatocytes (SC) representative images (the same tubule) are shown

To confirm these arrests of spermatogenesis, we inspected adult rat testicular sections for apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and revealed that Prdm9 KO/KO had a 7-fold higher mean number of apoptotic cells per all tubules in comparison to Prdm9 wt/wt and Prdm9 KO/wt (P < 0.001 for both cases LRM and Tukey’s test). To discern the germ cell types undergoing apoptosis, we combined these data with the results of anti-PIWIL1 immunohistochemistry (Fig. 6b, c). The PIWIL1 protein is expressed in pachytene spermatocytes and spermatids [32, 33]. Parallel analysis of 221 apoptotic tubules on neighboring TUNEL and anti-PIWIL1-hematoxylin-stained sections revealed that the majority of apoptotic cells (means of 82 to 88%, total 1059) were spermatocytes (P < 0.001 for both comparison of mutants to Prdm9 wt/wt and to Prdm9 KO/wt ). The mean number of apoptotic round spermatids per tubule in Prdm9 KO/KO also exceeded that in combined Prdm9 KO/wt and Prdm9 wt/wt (Fig. 6b, P = 0.0044), supporting the results of PAS-H staining. To further confirm the partial arrests of meiotic prophase I and metaphase, apoptotic cells were detected by the TUNEL assay on adult testicular sections co-labeled for acrosome (peanut agglutinin or PNA) and nucleus (Hoechst 33342) (Fig. 4b). Tubular stages IV and XIV in mutant testes carried more apoptotic cells (means of three testes 4.5 and 4.0%) than two wild-type controls (0.25 and 0.35%, respectively both P < 0.007, generalized linear model or GLM). The close-to-complete mid-pachytene arrest present in B6-Prdm9 KO/KO mutants [14, 34] is mild in the SHR mutants. Thus, the decreased sperm count in the Prdm9-deficient rats can be explained by increased apoptosis during at least three tubular stages, IV (mid-pachytene spermatocytes and round spermatids), XIV (metaphase spermatocytes), and V (round spermatids).

Rat PRDM9 is important for efficient spermiation

Mature rat spermatids (step 19 or S19) align at the luminal side of the seminiferous epithelium and are released into the tubular lumen during rat stage VIII in a process called spermiation [35]. To assess spermiation efficiency in Prdm9-deficient rats, we inspected the tubular stages IX and X from the rat testicular sections stained with PAS-H (Additional file 1: Fig. S4, Additional file 2: Table S3). Mature S19 wild-type SHR spermatids were rarely retained within the epithelium in stage IX (0.5 ± 0.4 per tubule), which contrasted with stage IX of Prdm9-deficient SHR (1.5 ± 0.2 P = 0.03, GLM). The S19 spermatid retention difference was also detected in wild-type versus mutant tubular stage X (0.2 ± 0.1 versus 1.8 ± 0.4 P = 0.01, GLM). These results suggested decreased spermiation efficiency. Thus increased apoptosis, abnormalities of round spermatids, and spermiation failure contribute to the reduced spermiogenic function of Prdm9-deficient rats. Altogether, our results show that PRDM9 is also required for normal rat haploid male gamete development and release.

Inactivation of Prdm9 leads to stage IV and XII arrests of PWD spermatocytes

To test the generality of two partial arrests of meiosis I at mid-pachytene and metaphase stages of Prdm9-deficient males, we used a Prdm9-deficient sterile PWD mouse male with gross reduction of sperm count [14]. We analyzed tubular stages IV and XII using PAS-H-stained testicular sections of adult Prdm9-deficient and control mouse PWD males (Additional file 1: Fig. S5, Additional file 2: Table S4). Comparing to wild-type PWD, we found increased numbers of degenerated pachytene spermatocytes in stage IV (4.9 ± 0.5 versus 0.0 ± 0.0 P < 0.001, GLM) and of metaphase cells in stage XII (3.2 ± 0.6 versus 0.7 ± 0.4 P < 0.001), suggesting that the two types of meiotic arrest reduce the numbers of Prdm9-deficient male germ cells.

Adult Prdm9-deficient PWD males display only 41% of wild-type pachytene spermatocytes with complete autosomal synapsis, while the rat SHR-Prdm9 KO/KO mutants display 72% of them, suggesting a weaker stage IV arrest in the rat. Consequently, we expected lower numbers of late primary spermatocytes in mouse tubular stage XII than in the corresponding rat stage XIV. Indeed, despite that the mean numbers of spermatogenic cells of all types in wild-type PWD mouse stage XII versus wild-type SHR rat stage XIV were similar, the mean counts of late primary spermatocytes were lower in the PWD mutant compared to the SHR mutant (4.1 ± 0.3 versus 7.7 ± 0.4), supporting the stronger mid-pachytene arrest in the PWD-Prdm9 tm/tm mutant. The sperm counts of the SHR and PWD mutants are about 33% and 1% of their corresponding wild-type counts. In agreement with these results, the number of elongated spermatids in stage IV from the SHR rat mutant were 17 to 37% and from the PWD mutant 0.3 to 3.9% of the SHR and PWD wild-type counts, respectively. In summary, both SHR rat and PWD mouse Prdm9-deficient males experience incomplete mid-pachytene and metaphase arrests.

Most but not all rat Prdm9-deficient spermatocytes synapse homologous chromosomes

As the B6-Prdm9 KO/KO mouse male displays complete and PWD-Prdm9 KO/KO partial pachytene arrest with persistent DSBs and incomplete synapsis [14, 34], we assayed adult rat pachytene nuclei by immunolabeling of the central element of the synaptonemal complex, the SYCP1 protein, and the DSB marker γH2AX (Fig. 7). The synaptonemal complex provides a scaffold for the alignment of homologous chromosomes and the central element keeps the homologs together through their entire length (homologous synapsis). Phosphorylated histone H2AX (γH2AX) is found in the vicinity of DSBs, but it also localizes to the X and Y chromosomes (sex body) at later meiotic stages (reviewed in [1]). Unlike Prdm9-deficient PWD males that carry 40% of normal (complete synapsis) pachytene spermatocytes [14], rat mutants displayed 78% (12 males, 729 nuclei) and wild-type SHR rats 96% normal pachytene spermatocytes (Fig. 7a). Four of 12 Prdm9-deficient males had a deletion of 20 codons in the PR/SET domain (516-bp deletion of total genomic DNA) and 72% normal pachynema while the other eight carried only truncating frameshift mutations (80%). There were no significant differences in the percentage of normal pachytene cells with fully synapsed autosomes between these four versus eight Prdm9-deficient rats (P = 0.09, GLM). Consistently, the testicular weights and sperm counts of these two Prdm9 deletion types were also similar (Fig. 2). To confirm the role of rat PRDM9 in synapsis, chromosome spreads from adult testes were also labeled using antibodies against the HORMAD2 protein that decorates asynapsed homologous chromosomes during pachynema ([36] Fig. 7b). Ninety-five percent of pachytene spermatocytes from Prdm9 KO/wt but only 62% from SHR-Prdm9 KO/KO animals were normal (P < 0.0001, GLM). Thus, rat SHR males require PRDM9 for efficient synapsis, but to a lesser degree than mouse PWD males and unlike B6 males, where PRDM9 is nearly essential for synapsis [14].

Decreased homologous synapsis but similar crossover rate in SHR males with Prdm9 deletions compared to wild-type. a, b Percentage of normal pachytene spermatocytes (with all autosomes synapsed). Each dot represents a single animal (over 50 cells). Antibodies used for the staining of chromosomal spreads are given in the headings of each graph. Nuclear spreads used for the analysis of autosomal synapsis in both a and b were used for the analysis of XY synapsis in c and d. c Percentage of nuclei with full autosomal synapsis out of nuclei with XY asynapsis in Prdm9-deficient and control rat testes. d percentage of nuclei with both autosomal and XY asynapsis in Prdm9-deficient and control rat testes. e, f, Examples of normal pachytene cells. Immunocytochemistry with antibodies against: e Synaptonemal complex (SYCP1, SYCP3) and chromatin surrounding DSB sites (γH2AX) f SYCP3, γH2AX, and unsynapsed chromosomal axes (HORMAD2). See Additional file 1: Fig. S6 for the images of representative nuclei from Prdm9-deficient males. Differences between Prdm9 genotypes were analyzed using LRM with subsequent Tukey test and Holm adjustment for multiple testing. g Representative image of chromosomal spread from a Prdm9-deficient rat immunostained for cyclin-dependent kinase 2 (CDK2) and synaptonemal complex (SYCP1), confirming that CDK2 localizes both to crossover nodules and telomeres as in the mouse. h Counts of autosomal crossover nodules per cell (N = 2 animals for both Prdm9 KO/wt and Prdm9 KO/KO , N = 1 for wild-type)

Rat PRDM9 is important for efficient synapsis of sex chromosomes

To check whether PRDM9 also supports synapsis of rat sex chromosomes, we analyzed spread nuclei of pachytene spermatocytes from adult testes using immunolabeling (Fig. 7c, d). Seven mutant testes carried 192 of 549 (35%) and seven controls (3 wild-type and 4 heterozygous Prdm9 males) 42 of 803 (5%) cells with XY asynapsis. Eighty-six percent (36 of 42) of control cells but only 35% (68 of 192 Fig. 7c) mutant cells displayed complete autosomal synapsis, thus probably being late pachynema. The remaining 124 Prdm9-deficient spermatocytes (23% of 549, 65% of 192) with both sex and autosomal asynapses were rare in the controls (0.7% of 803, 14% of 42 Fig. 7d) and represented aberrant cells. These results suggest that rat Prdm9 is required for efficient XY synapsis.

Rat PRDM9 supports repair of DSBs and does not affect the crossover rate

Mouse PRDM9 is important for efficient DSB repair [12, 34]. To assess the effect of PRDM9 on repair of meiotic DSBs in the rat, we analyzed spread testicular nuclei from Prdm9-deficient adult spermatocytes. Immunostaining for synaptonemal complex, centromeres, and early stages of DSB repair (RAD51/DMC1) revealed a few RAD51/DMC1 foci on autosomes and multiple foci on sex chromosomes in normal early pachytene spermatocytes (Additional file 1: Fig. S6m-p). All RAD51/DMC1 signal disappeared in normal late pachynema (Fig. S6q). However, abnormal cells displayed many strong RAD51/DMC1-stained foci on asynapsed chromosomes (Fig. S6r), suggesting that PRDM9 is important for efficient repair of programmed DSBs to crossovers.

Because the sperm presence in the mouse lacking Prdm9 function correlates with crossover rate [14], we analyzed the number of meiotic crossovers in rats with different Prdm9 copy numbers, including SHR-Prdm9 wt/wt and SHR-Prdm9 KO/KO males. Spermatocytes were immunostained with antibodies to the CDK2 protein that localizes to crossover sites in mouse meiotic prophase ([37] Fig. 7g). All Prdm9-deficient spermatocytes with SYCP1 and CDK2 signals found (63 from two males) were fully synapsed. Both Prdm9-deficient males tested carried on average 27.4 autosomal crossovers per cell, the same as three control males (27.4 ± 0.2 Fig. 7h), indicating no effect of Prdm9 on the mean crossover rate of rat males.

The reduced fertility parameters of Prdm9-deficient rats change with age

Prdm9 affects delayed fertility of intersubspecific mouse hybrid males [6]. To uncover the age-dependent effects of Prdm9 on rat fertility, we analyzed various fertility parameters at multiple time points up to 250 dpp. The Prdm9-deficient animals displayed lower fertility parameters at all ages. Except for body weight used as negative control, all fertility parameters tested started to decline in SHR-Prdm9 KO/KO but not in control animals after about 100–150 dpp (Fig. 8).

Fertility parameters of Prdm9-deficient animals change with age. Differences between three Prdm9 genotypes were analyzed using LRM with subsequent Tukey tests and Holm adjustment in three age groups: 50–100, 100–150, and 150–250 dpp. P values illustrate differences between SHR-Prdm9 KO/wt and SHR-Prdm9 KO/KO rats. The comparison of wild-type and SHR-Prdm9 KO/wt rats revealed no differences in fertility parameters and had highly consistent correlation curves. The correlation between age and body weight was used as a negative control

To assess male fertility more directly, we crossed the rat males and analyzed the offspring production (Fig. 2e, f). In agreement with their testicular weight and sperm counts, the Prdm9-deficient males sired less pups after 150 dpp, thus validating the effect of PRDM9 on age-dependent fertility.

Inactivation of rat Prdm9 leads to premature ovarian failure

PWD and B6 Prdm9-deficient mouse females are sterile, as they form very few follicles and lose all oocytes before adulthood [12, 14]. To explain the decreased ovarian weight of the Prdm9-deficient rats, we checked the effect of Prdm9 on follicular development and age of fertility. Folliculogenesis starts with formation of rat primordial follicles on 2 dpp. Our analysis revealed 25% primordial follicles in SHR-Prdm9 KO/KO compared to control females at 2 dpp (Fig. 3f), indicating that the majority of oocytes are lost before the onset of follicular development. Primary, secondary, and small antral follicles can be observed beside primordial follicles in juvenile rats before the onset of estrus on 21 dpp. The mean total number of follicles in the mutant was only 7% of controls at 21 dpp and almost no primordial follicles were detected. This decreased size of the oocyte pool likely contributes to a decreased littersize. Prdm9-deficient rats contained no follicles at 180 dpp, which contrasted with the controls (Fig. 3f, g). The paucity of oocytes explains the lack of offspring in mutant females after 150 dpp (Fig. 3d), despite being mated at this age (Fig. 3e), and it is also reflected by the decreasing ovarian weight (see above). These phenotypes of the SHR-Prdm9 KO/KO females resemble the human Premature Ovarian Failure syndrome, where the oocyte pool is exhausted early in age leading to sterility [38]. Altogether, these results suggest the importance of rat Prdm9 for oocyte survival and follicle development.

Rat PRDM9 affects the duration of meiotic prophase I in both sexes

Partial meiotic arrest in semifertile mouse hybrids, which is alleviated by manipulating Prdm9 dosage, is accompanied by meiotic delay [6] and SHR-Prdm9 KO/KO adult males displayed increased leptotene counts compared to controls in several tubular stages. To validate the requirement of Prdm9 for normal meiotic progression kinetics in SHR males, we analyzed the first wave of rat spermatogenesis by immunohisto- and cytochemistry. We inspected sections of prepubertal PRDM9-deficient and control (wild-type and Prdm9 KO/wt ) SHR testes immunolabeled for chromatin surrounding DNA breaks under repair (γH2AX) and cytoplasmic piwiRNA particles (PIWIL1). Fifty-two percent of testicular tubules in 21-dpp controls (3 males, 154 to 368 tubules scored) but only 8% tubules of their mutant littermates (565 tubules from two animals) were classified as containing cells in pachynema (Fig. 9a, b P = 0.042).

Delayed meiosis in male and female SHR rats lacking Prdm9 function. a-d Delayed prophase I in Prdm9-deficient male rats compared to controls. a, b Immunohistochemistry of 21-dpp testes (154 to 368 tubules scored from each animal). c Immunocytochemistry of 18-dpp testicular spreads (over 400 spermatocytes for each genotype). d Delayed meiosis is associated with apoptosis. Immunohistochemistry of prepubertal testes using TUNEL and DAPI. e Delayed onset of pachynema in Prdm9-deficient female rats. Spread 21-dpc ovarian nuclei were staged using immunocytochemistry (over 250 oocytes scored for each of the three Prdm9 genotypes LRM). The data underlying all published plots are in Additional file 3

To confirm the meiotic delay found at the tissue level also on cells, we immunostained spreads of testicular nuclei from 18-dpp males for synaptonemal complex markers. Pachytene spermatocytes formed about 8% of all spermatocytes in the mutant testes compared to 21% in controls (Fig. 9c). To evaluate the association of the mutant rat meiotic prophase I delay with partial meiotic arrest, we inspected testicular sections from 21-dpp SHR males for apoptosis using TUNEL staining. There was a higher percentage of tubules containing multiple apoptotic cells in the mutant compared to control testes (means of 24 versus 13%), suggesting a weak arrest of prophase I (Fig. 9d). This conclusion is supported by the analysis of PAS-H staining and apoptosis in tubular stage IV of adult rat mutant testes (see above).

Both male and female Prdm9-deficient SHR animals displayed age-dependent fertility. To evaluate the importance of PRDM9 for the timely meiotic progression in SHR females, we staged spread nuclei from embryonic ovaries, where the single wave of female meiotic prophase I takes place (Fig. 9e). We used markers of synaptonemal complex (SYCP3 and SYCP1) and chromatin surrounding DNA breaks under repair (γH2AX). Seven to 15% oocytes in four pairs of control ovaries at 21 days post coitum (dpc) were pachytene. However, two pairs of littermate mutant ovaries contained no pachytene nuclei (Fig. 9e P = 0.015, LRM), validating the prophase I delay of female rat meiosis in the absence of PRDM9.

Our results show that PRDM9 deficiency delays the onset of pachytene stage and slows down the progression of meiotic prophase I in SHR rats of both sexes.

Rat PRDM9 trimethylates H3K4 and guides most meiotic DSBs

In order to assess the function of rat PRDM9 in DSB positioning, genome-wide distributions of DSBs were analyzed in two related rat strains carrying the same Prdm9 allele, WKY and SHR, and in the BN/RIJHsd strain that harbors a distinct Prdm9 allele (Fig. 10, right) by single-stranded DMC1-bound DNA sequencing (SSDS). The identified DSB hotspots overlapped by 88% between the SHR and WKY strains, but only by 3% between the BN and WKY strains (Fig. 10, Additional file 1: Fig. S7). DSB hotspots were stronger on chromosome X than on autosomes (Additional file 1: Fig. S8), as expected [15]. The strain overlap data suggest that PRDM9 controls most sites of rat recombination initiation. To address the epigenetic function of rat PRDM9, we generated H3K4me3 profiles from the BN and WKY testes. The profiles differed at variable hotspot sites (Fig. 10), thus validating that rat PRDM9 displays in vivo H3K4-methyltransferase activity. Relocation of meiotic DSBs to H3K4me3 at promoters and other functional genomic elements in the Prdm9 knockout B6 mice was suggested as a possible cause of infertility. The alleviation of the infertile phenotype in the Prdm9-deficient rats could therefore be caused by the existence of another factor directing the recombination machineries away from these sites. The existence of this factor was hypothesized based on the analysis of the sites of PRDM9-independent human crossovers [23]. To check this possibility, genome-wide distribution of meiotic DSBs was compared between an adult SHR male carrying the homozygous KO39 truncating deletion (Fig. 1) and its wild-type littermate. The distribution of DSB hotspots in rats lacking PRDM9 was similar to that in mice: a large percentage of hotspots coincided with the default H3K4me3 marks, many of which were at gene promoters (Fig. 10 and see below). The relocation of DSB hotspots in the Prdm9-deficient rat to the default H3K4me3 sites (98.6% of 21,586 SHR DSB hotspots) and the similarity of the phenotypes of the four rat Prdm9 deletions support the view that the truncating mutations result in null alleles. Therefore, it is unlikely that the rat has other factors besides PRDM9 to avoid recombination at default H3K4me3 sites.

The positions of the rat meiotic DSB hotspots are determined by PRDM9. Left, a region of rat Chromosome 17 (rn5 assembly) exemplifying that the DSB hotspots (shown as peaks) in wild-type SHR males (SHR Prdm9 +/+ SSDS) are shared with the strain with the same Prdm9 allele (WKY Prdm9 +/+ SSDS), but not with the strain with different Prdm9 allele (BN/RIJHsd Prdm9 +/+ SSDS) or inactivated Prdm9 allele (SHR Prdm9 −/− SSDS). DSB hotspots in Prdm9-deficient male co-localize with testicular (T) and less with liver (L) H3K4me3 marks. The H3K4me3 profiles from the BN a WKY strains are different and reflect hotspot sites unique to each of these strains, thus validating that rat PRDM9 displays H3-methyltransferase activity. Some promoters correspond to Prdm9-independent hotspots (green arrowheads), but other promoters (pink arrowheads) are not targeted. The row “Genes” shows Ensembl gene models. Raw ChIP-seq coverage is shown in 150-bp windows each panel is scaled to the maximum value. Right, DNA-binding zinc-finger arrays of PRDM9 from two rat strains showing polymorphic amino acid residues in contact with DNA

Prdm9-independent hotspots tend to localize to orthologous regions

To learn about the evolutionary and functional properties of DSB hotspots (HSs), we analyzed the conservation of rat (SHR) and mouse (B6) HSs by mapping the rat HSs to the mouse genome assembly (see the “Methods” section). As expected from the analysis done in the mouse [14, 15], overlaps between wild-type HSs and Prdm9 KO/KO HSs were only 1 to 2% between the species (Fig. 11a). However, 40% of B6-Prdm9 KO/KO HSs corresponded to 37% of SHR-Prdm9 KO/KO HSs (Fig. 11a). Therefore, the recombination initiation maps were similar in the two PRDM9-deficient rodent species. This is analogous to different species in finches and budding yeast that naturally lack Prdm9 [17, 18].

Overlaps of recombination initiation hotspots from Prdm9-deficient rodents with a wild-type hotspots and b regulatory elements. c Overlaps of promoters with hotspots

In general, 36% (11,888 of 33,660) of rat and 41% (12,716 of 30,929) of mouse Prdm9-independent HSs localized to evidence-based promoters (defined by Ensembl, see the “Methods” section), in contrast to wild-type HSs from SHR rats (343 of 19,967 or 2%) or B6 mice (900 of 19,528 or 3%). Our analysis revealed that 66% of conserved Prdm9-independent HSs overlapped promoters, in contrast to 25% of mouse-specific and 18% of rat-specific Prdm9-independent HSs (Fig. 11b). When the set of promoters was narrowed to promoters of coding genes, 27% (8229 of 30,929) of mouse HSs and 23% (7687 of 33,360) of rat HSs fell within their coordinates.

The general analysis of promoters of coding genes revealed that 65% (14,237 of 21,956) of mouse promoters overlapped with 73% (14,447 of 19,719) of rat promoters (see the “Methods” section). Sixty-eight percent of the overlapping promoters contained HSs in either one or both rodent species, and 43% of conserved promoters overlapped conserved hotspots (Fig. 11c).


Gamete formation

A short review of mitosis, meiosis and cell differentiation begin this lesson which quickly moves on to outline the details of the processes of gametogenesis and spermatogenesis. In two final activities students make comparisons to learn the similarities and differences between these two processes.What type of cells are formed by meiosis?How many daughter cells are there after meiosis?How many chromosomes are there in.

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Gametogenesis (Spermatogenesis and Oogenesis)

Gametogenesis, the production of sperm and eggs, takes place through the process of meiosis. During meiosis, two cell divisions separate the paired chromosomes in the nucleus and then separate the chromatids that were made during an earlier stage of the cell’s life cycle. Meiosis produces haploid cells with half of each pair of chromosomes normally found in diploid cells. The production of sperm is called spermatogenesis and the production of eggs is called oogenesis .


Types of Gametogenesis: Spermatogenesis and Oogenesis | Biology

Gametogenesis is the process of formation and differentiation of haploid gametes (sperms and ova) from the diploid primary germ cells, gametogonia (spermatogonia and oogonia) present in primary sex organs called gonads (testes in male and ovaries in female respectively).

Gametogenesis is of two types:

I. Spermatogenesis (Figs. 3.13 A and 3.14):

Definition:

It is the formation of haploid, microscopic and functional male gametes, spermatozoa from the diploid reproductive cells, spermatogonia, present in the testes of male organism.

Period:

In the seasonally breeding animals, the testes undergo testicular cycle in which the testes and their spermatogenic tissue become functional only in the specific breeding season. So in some seasonally breeding mammals like bat, otter and llama, testes enlarge, become functional and descend into the scrotum in the breeding season as become heavier due to accumulation of sperms, while become reduced, non-functional and ascend into the abdomen in other seasons.

But in human male, lion, bull, horse etc., the testes lie permanently in the scrotum and spermatogenesis occurs throughout the year. In human male, testes descend into the respective scrotal sacs during seventh month of development under the stimulation of FSH of adenohypophysis.

But in some mammals e.g. elephant, echidna, dolphin, whale, seal etc., testes lie permanently in the abdomen (intra­-abdominal) mainly due to presence of blubber (thick fatty layer beneath the skin). Spermatogenesis is a continuous process and is completed in about 74 days.

Mechanism:

Spermatogenesis is divided into two parts:

A. Formation of Spermatid:

It is divided into three phases:

1. Multiplicative or Mitotic phase:

It involves the rapid mitotic division of diploid primary or primordial germ cells, called gonocytes, present in germinal epithelium of the seminiferous tubules of the testes. These cell are undifferentiated and have large and chromatin-rich nucleus.

This forms large number of diploid and rounded sperm mother cells called spermatogonia (Gr. sperma = seed gone = offspring). Each spermatogonial cell is about 12 pm in diameter and has a prominent nucleus. Some spermatogonia act as stem cells (called Type A spermatogonia) and go on dividing and adding new cells by repeated mitotic divisions, so forming spermatogenic lineage, but some spermatogonia move inward and enter growth phase (called Type B spermatogonia).

It is characterized by spermatocytogenesis in which a diploid spermatogonium increases in size (about twice) by the accumulation of nutritive materials (derived from germinal cells and not synthesized) in the cytoplasm and replication of DNA, and forms diploid primary spermatocyte. Nutritive materials are derived from germinal cells. During this, the primary spermatocyte prepares itself to enter meiosis. Growth phase of spermatogenesis is of much shorter duration than that of oogenesis.

3. Maturation or Meiotic phase:

It is characterised by meiosis. The diploid primary spermatocyte undergoes meiosis-I (reductional or heterotypical division) and forms two haploid cells called secondary spermatocytes, each containing 23 chromosomes.

It is immediately followed by meiosis-II (equational or homotypical division) in each secondary spermatocyte to form two haploid spermatids, each of which has 23 chromosomes. So each diploid spermatogonium produces 4 haploid spermatids. Different stages of spermatogenesis are interconnected by cytoplasmic strands till spermiogenesis when the maturing and interconnected gametes separate from each other.

B. Spermiogenesis (Fig. 3.15):

The transformation of a non-motile, rounded and haploid spermatid into a functional and motile spermatozoan is called spermiogenesis or spermioteliosis. The main aim is to increase the sperm motility by reducing weight and development of locomotory structure.

It involves the following changes:

1. Nucleus becomes condensed, narrow and anteriorly pointed due to loss of materials like RNAs, nucleolus and most of acidic proteins.

2. A part of Golgi body of spermatid forms the acrosome, while the lost part of Golgi body is called Golgi rest.

3. Centrioles of spermatid form the neck of sperm.

4. Distal centriole gives rise to axoneme.

5. Mitochondria form a spiral ring behind the neck around the distal centriole and proximal part of axoneme. This is called nebenkern.

6. Most of cytoplasm is lost but some cytoplasm forms sheath of tail of sperm.

The spermatids mature into spermatozoa in deep folds of the cytoplasm of the Sertoli cells (nurse cells) which also provide nourishment to them. Mature spermatozoa are released in the lumen of seminiferous tubules, called spermiation. The two testes of young adult form about 120 million sperms each day.

Changes in spermatid to form sperm during spermiogenesis.

Structure of spermatid

Forms axial filament of sperm tail.

Form mitochondrial spiral of sheath called nebenkem.

Generally lost except a thin sheath called manchette.

Control:

In human male, spermatogenesis starts only at the age of puberty due to increased secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus of brain. GnRH stimulates adenohypophysis to secrete two gonadotropins: FSH and ICSH. ICSH stimulates the Leydig’s cells of testis to secrete male sex hormones, called androgens, most important of which is testosterone.

Testosterone stimulates the spermatogenesis especially spermiogenesis. FSH stimulates the Sertoli cells of testis to secrete certain factors which helps in the process of spermatogenesis. It is called physiological control.

Types:

In man and a large number of other animals having XY mechanism in male, there are two types of sperms: 50% Gynosperms having X-Chromosome and 50’X) Androsperms having Y-Chromosome.

Significance:

(a) Produces haploid sperms.

(b) Crossing over may occur during meiosis-I, so producing variations.

(c) Proves evolutionary relationship.

II. Oogenesis (Fig. 3.13 B):

Definition:

It involves the formation of haploid female gametes called ova, from the diploid egg mother cells, oogonia, of ovary of female organism. It involves 2 biological processes: Genetical programming and packaging.

Period:

Period of oogenesis is different in different animals. In human female, there are about 1,700 primary germ cells in the undifferentiated female gonad at one month of foetal development. These proliferate to form about 600,000 oogonia at two months of gestation period and by its 5th month, the ovaries contain over 7 million oogonia however, many undergo atresia (degeneration of germ cells) before birth. At the time of birth, there are 2 million primary follicles, but 50% of these are atretic.

Atresia continues and at the time of puberty each ovary contains only 60,000-80,000 primary follicles. Oogenesis is completed only after the onset of puberty and only one out of 500 is stimulated by FSH to mature. So oogenesis is a discontinuous and wasteful process.

Mechanism:

Like the spermatogenesis, oogenesis is formed of three phases:

1. Multiplicative phase:

In this certain primary germ cells (larger in size and having large nuclei) of germinal epithelium of ovary undergo rapid mitotic divisions to form groups of diploid egg mother cells, oogonia. Each group is initially a chord and is called egg tube of pfluger which later forms a rounded mass, egg nest (Fig. 3.13 B).

Growth phase of oogenesis is of very long duration than that of spermatogenesis e.g., only three days in Drosophila, 6-14 days in hen, 3 years in frog and many years (12-13 years) in human female. During growth phase, one oogonium of egg nest is transformed into diploid primary oocyte while other oogonia of the egg nest form a single-layered nutritive follicular epithelium around it.

The structure so formed is called primary follicle. Later, each primary follicle gets surrounded by more layers of granulosal cells and changes into secondary follicle. Soon secondary follicle develops a fluid-filled antral cavity called antrum, and is called tertiary follicle. It further changes to form Graafian follicle. So not all the oogonia develop further.

Growth phase involves:

(a) Increase in size of oocyte (2000 times in frog 43 times in mouse 90,000 times in Drosophila 200 times in hen and about 200 times in human female) by the formation and accumulation of yolk (vitellogenesis) by a special mitochondrial cloud lying close to nucleus and called yolk nucleus.

(b) Nucleus becomes bloated with nucleoplasm and is called germinal vesicle.

(c) A thin vitelline membrane is secreted around the oocyte.

(d) Increase in number of mitochondria, amount of ER and Golgi body.

(e) Formation of lampbrush chromosomes in fishes, amphibians, reptiles, birds, insects, etc. for rapid yolk synthesis.

(f) Gene-amplification or redundancy of r-RNA genes for rapid synthesis of r-RNA.

It is characterized by meiosis. In this, the diploid and fully grown primary oocyte undergoes meiosis-I (reductional division) to form two unequal haploid cells. The smaller cell is called first polar body (Polocyte) and has very small amount of cytoplasm. The larger cell is called secondary oocyte and has bulk of nutrient-rich cytoplasm. Both of these are haploids and each has 23 chromosomes.

Secondary oocyte undergoes meiosis-II (equational division) to form two unequal haploid cells. The smaller cell is called second polar body and has very little of cytoplasm, while the larger cell is called ootid. It has almost whole of cytoplasm and differentiates into an ovum. Meanwhile, first polar body may divide into two.

So in oogenesis, a diploid oogonium forms one haploid ovum and two or three polar bodies while in spermatogenesis, a diploid spermatogonium forms four haploid sperms. The primary function of formation of polar bodies is to bring haploidy but to retain the whole of the cytoplasm in one ovum to provide food during the development of zygote to form an embryo. The number of ova is reduced with the ability of the female to bear and rear them.

In most of organisms including human female, the ovulation occurs at secondary oocyte stage in which meiosis-I has been completed and first polar body has been released. Meiosis-II is completed only at the time of sperm-entry.


228 Human Reproductive Anatomy and Gametogenesis

By the end of this section, you will be able to do the following:

  • Describe human male and female reproductive anatomies
  • Discuss the human sexual response
  • Describe spermatogenesis and oogenesis and discuss their differences and similarities

As animals became more complex, specific organs and organ systems developed to support specific functions for the organism. The reproductive structures that evolved in land animals allow males and females to mate, fertilize internally, and support the growth and development of offspring.

Human Reproductive Anatomy

The reproductive tissues of male and female humans develop similarly in utero until a low level of the hormone testosterone is released from male gonads. Testosterone causes the undeveloped tissues to differentiate into male sexual organs. When testosterone is absent, the tissues develop into female sexual tissues. Primitive gonads become testes or ovaries. Tissues that produce a penis in males produce a clitoris in females. The tissue that will become the scrotum in a male becomes the labia in a female that is, they are homologous structures.

Male Reproductive Anatomy

In the male reproductive system, the scrotum houses the testicles or testes (singular: testis), including providing passage for blood vessels, nerves, and muscles related to testicular function. The testes are a pair of male reproductive organs that produce sperm and some reproductive hormones. Each testis is approximately 2.5 by 3.8 cm (1.5 by 1 in.) in size and divided into wedge-shaped lobules by connective tissue called septa. Coiled in each wedge are seminiferous tubules that produce sperm.

Sperm are immobile at body temperature therefore, the scrotum and penis are external to the body, as illustrated in (Figure) so that a proper temperature is maintained for motility. In land mammals, the pair of testes must be suspended outside the body at about 2 ° C lower than body temperature to produce viable sperm. Infertility can occur in land mammals when the testes do not descend through the abdominal cavity during fetal development.


Which of the following statements about the male reproductive system is false?

  1. The vas deferens carries sperm from the testes to the penis.
  2. Sperm mature in seminiferous tubules in the testes.
  3. Both the prostate and the bulbourethral glands produce components of the semen.
  4. The prostate gland is located in the testes.

Sperm mature in seminiferous tubules that are coiled inside the testes, as illustrated in (Figure). The walls of the seminiferous tubules are made up of the developing sperm cells, with the least developed sperm at the periphery of the tubule and the fully developed sperm in the lumen. The sperm cells are mixed with “nursemaid” cells called Sertoli cells which protect the germ cells and promote their development. Other cells mixed in the wall of the tubules are the interstitial cells of Leydig. These cells produce high levels of testosterone once the male reaches adolescence.

When the sperm have developed flagella and are nearly mature, they leave the testicles and enter the epididymis, shown in (Figure). This structure resembles a comma and lies along the top and posterior portion of the testes it is the site of sperm maturation. The sperm leave the epididymis and enter the vas deferens (or ductus deferens), which carries the sperm, behind the bladder, and forms the ejaculatory duct with the duct from the seminal vesicles. During a vasectomy, a section of the vas deferens is removed, preventing sperm from being passed out of the body during ejaculation and preventing fertilization.

Semen is a mixture of sperm and spermatic duct secretions (about 10 percent of the total) and fluids from accessory glands that contribute most of the semen’s volume. Sperm are haploid cells, consisting of a flagellum as a tail, a neck that contains the cell’s energy-producing mitochondria, and a head that contains the genetic material. (Figure) shows a micrograph of human sperm as well as a diagram of the parts of the sperm. An acrosome is found at the top of the head of the sperm. This structure contains lysosomal enzymes that can digest the protective coverings that surround the egg to help the sperm penetrate and fertilize the egg. An ejaculate will contain from two to five milliliters of fluid with from 50–120 million sperm per milliliter.


The bulk of the semen comes from the accessory glands associated with the male reproductive system. These are the seminal vesicles, the prostate gland, and the bulbourethral gland, all of which are illustrated in (Figure). The seminal vesicles are a pair of glands that lie along the posterior border of the urinary bladder. The glands make a solution that is thick, yellowish, and alkaline. As sperm are only motile in an alkaline environment, a basic pH is important to reverse the acidity of the vaginal environment. The solution also contains mucus, fructose (a sperm mitochondrial nutrient), a coagulating enzyme, ascorbic acid, and local-acting hormones called prostaglandins. The seminal vesicle glands account for 60 percent of the bulk of semen.

The penis , illustrated in (Figure), is an organ that drains urine from the renal bladder and functions as a copulatory organ during intercourse. The penis contains three tubes of erectile tissue running through the length of the organ. These consist of a pair of tubes on the dorsal side, called the corpus cavernosum, and a single tube of tissue on the ventral side, called the corpus spongiosum. This tissue will become engorged with blood, becoming erect and hard, in preparation for intercourse. The organ is inserted into the vagina culminating with an ejaculation. During intercourse, the smooth muscle sphincters at the opening to the renal bladder close and prevent urine from entering the penis. An orgasm is a two-stage process: first, glands and accessory organs connected to the testes contract, then semen (containing sperm) is expelled through the urethra during ejaculation. After intercourse, the blood drains from the erectile tissue and the penis becomes flaccid.

The walnut-shaped prostate gland surrounds the urethra, the connection to the urinary bladder. It has a series of short ducts that directly connect to the urethra. The gland is a mixture of smooth muscle and glandular tissue. The muscle provides much of the force needed for ejaculation to occur. The glandular tissue makes a thin, milky fluid that contains citrate (a nutrient), enzymes, and prostate specific antigen (PSA). PSA is a proteolytic enzyme that helps to liquefy the ejaculate several minutes after release from the male. Prostate gland secretions account for about 30 percent of the bulk of semen.

The bulbourethral gland , or Cowper’s gland, releases its secretion prior to the release of the bulk of the semen. It neutralizes any acid residue in the urethra left over from urine. This usually accounts for a couple of drops of fluid in the total ejaculate and may contain a few sperm. Withdrawal of the penis from the vagina before ejaculation to prevent pregnancy may not work if sperm are present in the bulbourethral gland secretions. The location and functions of the male reproductive organs are summarized in (Figure).

Male Reproductive Anatomy
Organ Location Function
Scrotum External Carry and support testes
Penis External Deliver urine, copulating organ
Testes Internal Produce sperm and male hormones
Seminal Vesicles Internal Contribute to semen production
Prostate Gland Internal Contribute to semen production
Bulbourethral Glands Internal Clean urethra at ejaculation

Female Reproductive Anatomy

A number of reproductive structures are exterior to the female’s body. These include the breasts and the vulva, which consists of the mons pubis, clitoris, labia majora, labia minora, and the vestibular glands, all illustrated in (Figure). The location and functions of the female reproductive organs are summarized in (Figure). The vulva is an area associated with the vestibule which includes the structures found in the inguinal (groin) area of women. The mons pubis is a round, fatty area that overlies the pubic symphysis. The clitoris is a structure with erectile tissue that contains a large number of sensory nerves and serves as a source of stimulation during intercourse. The labia majora are a pair of elongated folds of tissue that run posterior from the mons pubis and enclose the other components of the vulva. The labia majora derive from the same tissue that produces the scrotum in a male. The labia minora are thin folds of tissue centrally located within the labia majora. These labia protect the openings to the vagina and urethra. The mons pubis and the anterior portion of the labia majora become covered with hair during adolescence the labia minora is hairless. The greater vestibular glands are found at the sides of the vaginal opening and provide lubrication during intercourse.


Female Reproductive Anatomy
Organ Location Function
Clitoris External Sensory organ
Mons pubis External Fatty area overlying pubic bone
Labia majora External Covers labia minora
Labia minora External Covers vestibule
Greater vestibular glands External Secrete mucus lubricate vagina
Breasts External Produce and deliver milk
Ovaries Internal Carry and develop eggs
Oviducts (Fallopian tubes) Internal Transport egg to uterus
Uterus Internal Support developing embryo
Vagina Internal Common tube for intercourse, birth canal, passing menstrual flow

The breasts consist of mammary glands and fat. The size of the breast is determined by the amount of fat deposited behind the gland. Each gland consists of 15 to 25 lobes that have ducts that empty at the nipple and that supply the nursing child with nutrient- and antibody-rich milk to aid development and protect the child.

Internal female reproductive structures include ovaries, oviducts, the uterus , and the vagina, shown in (Figure). The pair of ovaries is held in place in the abdominal cavity by a system of ligaments. Ovaries consist of a medulla and cortex: the medulla contains nerves and blood vessels to supply the cortex with nutrients and remove waste. The outer layers of cells of the cortex are the functional parts of the ovaries. The cortex is made up of follicular cells that surround eggs that develop during fetal development in utero. During the menstrual period, a batch of follicular cells develops and prepares the eggs for release. At ovulation, one follicle ruptures and one egg is released, as illustrated in (Figure)a.


The oviducts , or fallopian tubes, extend from the uterus in the lower abdominal cavity to the ovaries, but they are not in contact with the ovaries. The lateral ends of the oviducts flare out into a trumpet-like structure and have a fringe of finger-like projections called fimbriae, illustrated in (Figure)b. When an egg is released at ovulation, the fimbrae help the nonmotile egg enter into the tube and passage to the uterus. The walls of the oviducts are ciliated and are made up mostly of smooth muscle. The cilia beat toward the middle, and the smooth muscle contracts in the same direction, moving the egg toward the uterus. Fertilization usually takes place within the oviducts and the developing embryo is moved toward the uterus for development. It usually takes the egg or embryo a week to travel through the oviduct. Sterilization in women is called a tubal ligation it is analogous to a vasectomy in males in that the oviducts are severed and sealed.

The uterus is a structure about the size of a woman’s fist. This is lined with an endometrium rich in blood vessels and mucus glands. The uterus supports the developing embryo and fetus during gestation. The thickest portion of the wall of the uterus is made of smooth muscle. Contractions of the smooth muscle in the uterus aid in passing the baby through the vagina during labor. A portion of the lining of the uterus sloughs off during each menstrual period, and then builds up again in preparation for an implantation. Part of the uterus, called the cervix, protrudes into the top of the vagina. The cervix functions as the birth canal.

The vagina is a muscular tube that serves several purposes. It allows menstrual flow to leave the body. It is the receptacle for the penis during intercourse and the vessel for the delivery of offspring. It is lined by stratified squamous epithelial cells to protect the underlying tissue.

Sexual Response during Intercourse

The sexual response in humans is both psychological and physiological. Both sexes experience sexual arousal through psychological and physical stimulation. There are four phases of the sexual response. During phase one, called excitement, vasodilation leads to vasocongestion in erectile tissues in both men and women. The nipples, clitoris, labia, and penis engorge with blood and become enlarged. Vaginal secretions are released to lubricate the vagina to facilitate intercourse. During the second phase, called the plateau, stimulation continues, the outer third of the vaginal wall enlarges with blood, and breathing and heart rate increase.

During phase three, or orgasm, rhythmic, involuntary contractions of muscles occur in both sexes. In the male, the reproductive accessory glands and tubules constrict placing semen in the urethra, then the urethra contracts expelling the semen through the penis. In women, the uterus and vaginal muscles contract in waves that may last slightly less than a second each. During phase four, or resolution, the processes described in the first three phases reverse themselves and return to their normal state. Men experience a refractory period in which they cannot maintain an erection or ejaculate for a period of time ranging from minutes to hours.

Gametogenesis (Spermatogenesis and Oogenesis)

Gametogenesis, the production of sperm and eggs, takes place through the process of meiosis. During meiosis, two cell divisions separate the paired chromosomes in the nucleus and then separate the chromatids that were made during an earlier stage of the cell’s life cycle. Meiosis produces haploid cells with half of each pair of chromosomes normally found in diploid cells. The production of sperm is called spermatogenesis and the production of eggs is called oogenesis .

Spermatogenesis


Spermatogenesis, illustrated in (Figure), occurs in the wall of the seminiferous tubules ((Figure)), with stem cells at the periphery of the tube and the spermatozoa at the lumen of the tube. Immediately under the capsule of the tubule are diploid, undifferentiated cells. These stem cells, called spermatogonia (singular: spermatagonium), go through mitosis with one offspring going on to differentiate into a sperm cell and the other giving rise to the next generation of sperm.

Meiosis starts with a cell called a primary spermatocyte. At the end of the first meiotic division, a haploid cell is produced called a secondary spermatocyte. This cell is haploid and must go through another meiotic cell division. The cell produced at the end of meiosis is called a spermatid and when it reaches the lumen of the tubule and grows a flagellum, it is called a sperm cell. Four sperm result from each primary spermatocyte that goes through meiosis.

Stem cells are deposited during gestation and are present at birth through the beginning of adolescence, but in an inactive state. During adolescence, gonadotropic hormones from the anterior pituitary cause the activation of these cells and the production of viable sperm. This continues into old age.

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Oogenesis

Oogenesis, illustrated in (Figure), occurs in the outermost layers of the ovaries. As with sperm production, oogenesis starts with a germ cell, called an oogonium (plural: oogonia), but this cell undergoes mitosis to increase in number, eventually resulting in up to about one to two million cells in the embryo.


The cell starting meiosis is called a primary oocyte, as shown in (Figure). This cell will start the first meiotic division and be arrested in its progress in the first prophase stage. At the time of birth, all future eggs are in the prophase stage. At adolescence, anterior pituitary hormones cause the development of a number of follicles in an ovary. This results in the primary oocyte finishing the first meiotic division. The cell divides unequally, with most of the cellular material and organelles going to one cell, called a secondary oocyte, and only one set of chromosomes and a small amount of cytoplasm going to the other cell. This second cell is called a polar body and usually dies. A secondary meiotic arrest occurs, this time at the metaphase II stage. At ovulation, this secondary oocyte will be released and travel toward the uterus through the oviduct. If the secondary oocyte is fertilized, the cell continues through the meiosis II, producing a second polar body and a fertilized egg containing all 46 chromosomes of a human being, half of them coming from the sperm.

Egg production begins before birth, is arrested during meiosis until puberty, and then individual cells continue through at each menstrual cycle. One egg is produced from each meiotic process, with the extra chromosomes and chromatids going into polar bodies that degenerate and are reabsorbed by the body.

Section Summary

As animals became more complex, specific organs and organ systems developed to support specific functions for the organism. The reproductive structures that evolved in land animals allow males and females to mate, fertilize internally, and support the growth and development of offspring. Processes developed to produce reproductive cells that had exactly half the number of chromosomes of each parent so that new combinations would have the appropriate amount of genetic material. Gametogenesis, the production of sperm (spermatogenesis) and eggs (oogenesis), takes place through the process of meiosis.

(Figure) Which of the following statements about the male reproductive system is false?


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