First Wave Of Spermatogenesis Research Articles

Pituitary gonadotrophs are strongly activated at the beginning of spermatogenesis in African catfish, Clarias gariepinus.

Pituitary gonadotrophs were studied in male African catfish between 1 and 37 wk of age using antisera against the LH subunits for immunohistological and radioimmunological purposes, and cRNA probes for in situ hybridization. Immunoreactive material was already detectable at the earliest age examined. In juveniles, the signal for the common glycoprotein alpha subunit (GP alpha) was stronger than that for the LH beta subunit. Accordingly, an excess of radioimmunoassayable GP alpha 100 times that of LHbeta was recorded in the pituitary. Using in situ hybridization, the mRNAs were detected 7 (GP alpha) and 13 (LHbeta) wk after hatching. Detection of LHbeta mRNA coincided with a 300-fold increase in the pituitary content of LHbeta and intact LH, whereas GP alpha increased only 15-fold. The number of gonadotrophs per pituitary and the amount of LH per gonadotroph also increased strongly. The strong, initial increase in pituitary LH levels was always associated with the presence of spermatocytes. However, in a limited number of cases (3 out of 12 fish), the pituitary LH content was low despite the presence of spermatocytes. The number of gonadotrophs, the staining intensities (reflecting protein and mRNA), and the pituitary LH content kept increasing, although at a reduced rate, until completion of the first wave of spermatogenesis. In view of the excess of GP alpha over LHbeta, we conclude that expression of the two subunits is regulated in part by different mechanisms, and that expression of LHbeta is rate-limiting for the amount of intact LH. The strong activation of the gonadotrophs shortly after meiosis opens the possibility that a signal of testicular origin stimulates LH expression, in particular its beta subunit. In the absence of a FSH-like gonadotropin in catfish, we propose that LH covers all functions requiring gonadotropic regulation in the African catfish.

An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.

Morphological analysis of germ cell apoptosis during postnatal testis development in normal and Hsp 70-2 knockout mice.

The present study examined the occurrence of apoptotic cell death in the testis of wild-type mice from postnatal days 3 to 26 and in juvenile Hsp 70-2 knockout mice. Adult Hsp 70-2 knockout males are infertile and lack spermatids and spermatozoa (Dix et al. [1996a] Proc. Natl. Acad. Sci. U.S.A. 93:3264-3268). To identify the cell types undergoing apoptosis, we also examined the relationship between the occurrence of apoptotic cell death and the expression pattern of the Hsp 70-2 gene product (heat-shock protein 70-2 [HSP70-2]; marker for spermatocytes and spermatids), germ cell nuclear antigen 1 (GCNA1;marker for spermatogonia and spermatocytes), and vimentin (marker for Sertoli cells). This study shows that during postnatal development of the wildtype mouse testis (1) the percentage of apoptotic cell death detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method is higher in mice from days 8 to 22 than in younger or older mice, (2) the majority of apoptotic cells are spermatogonia and less frequently are spermatocytes, and (3) the degenerative cell death of spermatogonia and primary spermatocytes involves apoptosis with fragmentation of DNA. The analysis of apoptotic cell death in the testes of juvenile Hsp 70-2 knockout mice showed an additional increased level of apoptosis at day 17, during the first wave of spermatogenesis, in pachytene spermatocytes.

Differentiation-dependent enhanced expression of protein phosphatase 2Cβ in germ cells of mouse seminiferous tubules

The presence of five distinct isoforms of protein phosphatase 2Cβ (PP2C β1∼-5) is known. In this study, we demonstrate that the mRNA levels of PP2Cβ-3, -4 and -5 and PP2Cβ protein level increased during the course of the first wave of spermatogenesis in neonatal mouse testis. Northern blot and in situ hybridization analyses revealed that PP2Cβ-3, -4 and -5 were expressed predominantly in pachytene spermatocytes and in more highly differentiated germ cells. The substrate specificity of PP2Cβ-4 determined with artificial substrates differed from those of PP2Cβ-3 and -5, suggesting that the difference in the structure of PP2Cβ-3, -4 and -5 reflect their unique physiological functions in testicular germ cells.

Developmentally Regulated Expression ofHsp70-2and aHsp70-2/lacZTransgene during Spermatogenesis

Germ cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation ofHsp70-2,we used mRNA primer-extension, reverse transcriptase PCR (RT–PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5′-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression ofHsp70-2during spermatogenesis. Results with multiple lines of transgenic mice containing promoter–reporter transgenes with varying lengths ofHsp70-2sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required forHsp70-2/lacZexpression in spermatocytes. Histochemical detection of transgene β-galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the first wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution ofHsp70-2andHsp70-2/lacZmRNAs was determined by Northern blot,in situhybridization, and RT–PCR, and it was found that upregulation of expression of bothHsp70-2andHsp70-2/lacZwas specific to the meiotic phase of spermatogenesis.

Spontaneous Recrudescence of Spermatogenesis in the Photoinhibited Male Djungarian Hamster, Phodopus Sungorus1

Photosensitive rodents exposed to inhibitory short photoperiods become insensitive to this environmental factor after prolonged exposure. During the following process of spontaneous recrudescence, the animals that have adapted to the winter season show a return of all seasonal parameters. In the Djungarian hamster, obvious photoperiod-dependent changes are reinitiation of the reproductive organs, a 20-30% increase in body weight, and a moult from whitish fur into brown summer fur. This study was designed to analyze the morphological and endocrinological changes occurring during spontaneous testicular recrudescence in male Djungarian hamsters under prolonged short photoperiods. Two experiments were performed 1) to analyze the time-dependent changes in groups of hamsters exposed to short photoperiods and 2) to observe testicular and humoral changes in individual animals during spontaneous recrudescence. Regrowth of the testes and seminal vesicles did not begin before Week 18 in short photoperiods. While serum testosterone did not increase before Week 24, serum FSH had already returned to normal values from Week 18 onwards. Individual analysis by enzyme histochemistry revealed that 3 beta-hydroxysteroid-dehydrogenase activity in Leydig cells was not restored before testicular weights of more than 400 mg were observed and the first wave of spermatogenesis had reached the stage of elongated spermatids. This indicates that the testicular testosterone production was low until a status of testicular recrudescence had been achieved, at which point the testis showed complete qualitative spermatogenesis and a restoration of the Sertoli cell actin filaments. These data suggest that the process of early spontaneous recrudescence in male Djungarian hamsters appears to be initiated by the restoration of serum FSH rather than by testosterone.

Developmental stage- and spermatogenic cycle-specific expression of transcription factor GATA-1 in mouse Sertoli cells.

GATA-1 is an essential factor for the transcriptional activation of erythroid-specific genes, and is also abundantly expressed in a discrete subset of cells bordering the seminiferous epithelium in tubules of the murine testis. In examining normal and germ-line defective mutant mice, we show here that GATA-1 is expressed only in the Sertoli cell lineage in mouse testis. GATA-1 expression in Sertoli cells is induced concomitantly with the first wave of spermatogenesis, and GATA-1-positive cells are uniformly distributed among all tubules during prepubertal testis development. However, the number of GATA-1-positive cells declines thereafter and were found only in the peripheral zone of seminiferous tubules in stages VII, VIII and IX of spermatogenesis in the adult mouse testis. In contrast, virtually every Sertoli cell in mutant W/Wv, jsd/jsd or cryptorchid mice (all of which lack significant numbers of germ cells) expresses GATA-1, thus showing that the expression of this transcription factor is negatively controlled by the maturing germ cells. These observations suggest that transcription factor GATA-1 is a developmental stage- and spermatogenic cycle-specific regulator of gene expression in Sertoli cells.

Reduced testosterone during puberty results in a midspermiogenic lesion.

The aim of this study was to determine the role of testosterone, as reflected in the testicular interstitial fluid, in the completion of the first wave of spermatogenesis and to further elucidate its role in spermiogenesis. At weekly intervals beginning with 26-day-old rats, body and testis weights were obtained, testicular interstitial fluid testosterone (TIF-T) was assayed, daily sperm production (DSP) was determined, and testicular tissue was structurally analyzed by light and electron microscopy. At 40 days postpartum, half the rats were treated with ethane dimethanesulphonate (EDS) to temporarily reduce Leydig cells. The other half served as controls and were treated with the vehicle. The timing of EDS treatment was just prior to the elongation of spermatids. At Day 47 (1 week after EDS treatment), TIF-T, testis weight, DSP, and number of Leydig cells were significantly reduced. At Day 54 (2 weeks after treatment), TIF-T had returned to the normal adult level, Leydig cell repopulation was apparent, and testis weight was normal. The DSP returned to normal by Day 61 (3 weeks after treatment). At 1 and 2 weeks after treatment, Step 8-9 spermatids were partially or completely detached from Sertoli cells. Results indicate that a temporary reduction of testosterone during the peripubertal period leads to a temporary reduction of the DSP approximately 1 week later. It is suggested that reduced testosterone is associated with a mid-spermiogenic lesion interfering with stable attachment of Step 8-9 spermatids to Sertoli cells during Stage VIII-IX of the spermatogenic cycle.

Endocytic activity of male germ cells during puberty

It is well known that numerous germ cells degenerate during the first meiotic division and spermatid elongation is due mostly to an adverse physiological microenvironment in relation to hormonal deficiency. The present study is aimed at investigating the endocytic activity of germ cells, during the first wave of mouse spermatogenesis, using transferrin coupled to gold particles, in order to study the efficiency of this possible pathway of communication between Sertoli cells and germ cells. Labelling experiments in control animals confirmed a receptor-mediated pathway in all germ cells during puberty. Furthermore, our morphological and quantitative data revealed that during spermatid elongation, degenerating germ cells possessed a highly developed endocytic apparatus which contained twice as many transferrin gold particles than normal adult cells. The fact that endocytosis of transferrin was increased in degenerating germ cells indicates that, most probably, germ cell degeneration during the first wave of spermatogenesis did not result from a deficiency in iron transport. The higher endocytic activity of degenerating germ cells, compared to adult control cells, could not only be the result of a simple process of plasma membrane internalization but also a complex mechanism which could be involved in the degradation of the cells.

Expression of Basic Fibroblast Growth Factor-Like Substance in Immature Rat Testis: Immunocytochemical Localization and Detection of mRNA.

We have previously described immunoreactive basic fibroblast growth factor (bFGF)-like substance in adult rat testis and have shown that this bFGF-like substance presents in the nucleus of germ cells. In the present study, the distribution of bFGF was examined immunohistochemically in 3 or 12-day-old rat testes. In 3-day-old, immunoreactivity for bFGF was detected in the cytoplasm of gonocytes (prespermatogonia) and fetal Leydig cells. In contrast, in 12-day-old, immunoreactivity for bFGF was detected exclusively in the nucleus of germ cells. Thus, bFGF seems to translocate from the cytoplasm to the nucleus during formation of the first wave of spermatogenesis. Using reverse transcription-polymerase chain reaction (RT-PCR), bFGF mRNA was detected in the 3-day-old rat whole testis. Moreover, in situ hybridization analysis revealed gonocytes and fetal Leydig cells contain mRNA for bFGF. These results indicate that bFGF is synthesized in gonocytes and fetal Leydig cells before the formation of the first wave of spermatogenesis.

Polyamine profiles in rat testis, germ cells and Sertoli cells during testicular maturation.

Polyamine cellular concentrations (putrescine, spermidine and spermine) in the rat testis and testicular cell types were determined by fluorescence spectroscopy of their dansyl derivatives. A method is described to separate dansylated polyamines by high performance liquid chromatography in less than 12 minutes. In rat Sertoli cells, polyamine concentrations (per mg DNA) were greater than those in germ cells and the testis. The concentrations of all three polyamines increased with age. Concentrations of spermidine and spermine in germ cells also increased with age and leveled off after 27 to 35 days. On the other hand, higher putrescine levels were found in the testis of young rats (13 to 22 days) while the greatest spermidine and spermine contents were observed in the testis from rats of 31 to 35 days old. Of great interest, Sertoli cells from all age groups studied released a relatively large quantity of putrescine and a smaller amount of spermidine, but no spermine, into culture media. The amount of polyamine released by Sertoli cells varied with the age of the animal. Sertoli cells from 27-day-old rats released the greatest quantity of putrescine on a per mg DNA basis. The release of putrescine increased after hypotonic treatment that removed contaminating germ cells from the remaining Sertoli cells. It is concluded that cellular polyamine levels in the rat testis, germ cells and cultured Sertoli cells and the amount of polyamines released by Sertoli cells were age-dependent during the first wave of spermatogenesis.

The kinetics of the first wave of spermatogenesis in the newborn male mouse

AbstractA combination of autoradiography and air‐dried techniques was used to calculate the duration of the major meiotic stages in the first wave of spermatogenesis in the newborn mouse. The data indicated that the entry into meiosis occurred asynchronously over 2 days, and the time required for each stage and the total cycle was constant. These time intervals were nearly identical with those estimated for adult animals in the present study and by other authors.

Morphological and temporal sequence of meiotic prophase development at puberty in the male mouse.

The correct sequence of meiotic prophase development in the male mouse has been established by the use of pubertal males. The first wave of spermatogenesis at this time provides a unique opportunity to study progressive meiotic development in a direct way. Air-dried and micro-spread analyses have been carried out. Temporal and morphological progression at this time is entirely consistent with that occurring in the later waves of meiosis of the adult male. Morphological detail shows delayed pairing of the X and Y chromosomes relative to the autosomes. The longest XY synaptonemal complex is seen in early pachytene cells, occupying up to 72% of the length of the Y and 22% of the length of the X axis. By late pachytene, end-to-end pairing in the XY bivalent is established, the autosomal axes remaining fully paired. Desynapsis of the autosomes commences at early diplotene. A 'diffuse' diplotene stage in the male, comparable to the dictyate stage of the female, could not be found. Marked lengthening of the XY and autosomal axes did, however, occur through the diplotene stage.

Characterization of Sertoli cell RNA synthetic activities in vitro at selected times during sexual maturation.

In this study, Sertoli cell RNA synthetic activity in vitro was characterized at selected times during sexual maturation. Sertoli cells, isolated from rat testes undergoing the first wave of spermatogenesis and placed in culture for 4 days, exhibited 2-fold increases in soluble ribonucleotide pools and in total RNA concentrations over the age span of 18-35 days. High performance liquid chromatographic analysis of the ribonucleotide pools in Sertoli cells cultured from 18- and 33- to 34-day-old rats revealed that, in addition to the overall age-related doubling of concentrations, uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools were disproportionately increased 4- and 6-fold, respectively. In general, Sertoli cell contained relatively small amounts of UTP in comparison to several other cell types, but exhibited a high ADP:ATP ratio. A uniform 2-fold increase in the base composition of Sertoli cell RNA per mg DNA was observed over the age span of 18-35 days, with no preferential increase in any one specific nucleotide. There was no change in [3H]uridine incorporation (2 h) into RNA per cell (pmol/mg DNA), but decreased specific activity of the RNA (pmol/mg RNA) in Sertoli cells cultured from 35-day-old rats as compared to those from 18- to 19-day-old rats. Similar differences were noted in the specific activity of label incorporated into specific RNA bases. In contrast, the specific activity of the UTP-CTP soluble pool/mg DNA was only slightly increased. These data indicate that processes related to RNA synthesis in the Sertoli cell undergo a number of changes during the period of sexual maturation.

Control of spermatogenesis in primates: Effects of active immunization against FSH in the monkey

Spermatogenesis and steroidogenesis are 2 important functions of the testis. It is well known that the 2 pituitary hormones, namely FSH and LH, regulate these functions of the testis. Based on the early work of Greep et al. 1936; Greep & Fevold 1937), FSH is held to be responsible for control of spermatogenesis and LH for testosterone production. However, the precise role of FSH in spermatogenesis is not clear. We used specific and potent antisera to FSH to neutralize this hormone and study its role in spermatigenic function of the immature and adult rat (Madhwa & Dym 1976; Madhwa Raj et al. 1980). Our earlier work on the rat demonstrated the need for FSH during the first wave of spermatogenesis, but not in the adult rat. Further, the precise role of FSH in spermatogenesis of primates including the human male is also not clear. In view of this, we have undertaken studies to examine the effects of neutralizing FSH activity on spermatogenesis in the monkey, M fasicularies.

Glucose-6-phosphate dehydrogenase activity changes during spermatogenesis: Possible relevance to X-chromosome inactivation

Specific activities of glucose-6-phosphate dehydrogenase (G6PD) have been measured in mouse testicles during the first wave of spermatogenesis and in spermatogenic cells separated by the staput technique. The data are compatible with a premeiotic synthesis of spermatozoal G6PD. Thus, the presence of this X-linked gene product in spermatozoa seems not to be in conflict with the theory of X-inactivation during spermatogenesis.

Altered sexual development in male rats after oestrogen administration during the neonatal period.

Male rats given 250 mug oestradiol benzoate by subcutaneous injection on Day 4 of postnatal life showed a marked delay in the onset of the pubertal increase in the weight of the testes and seminal vesicles and in spermatogenesis but not a complete failure of sexual development. The increase in plasma testosterone concentration at puberty was also delayed in oestrogen-treated males but the eventual increase in seminal vesicle weight was closely related in time to the delayed increase in plasma testosterone concentration. Both plasma LH and FSH concentrations were reduced for about 10 days after oestrogen administration as compared to control values. After 22 days of age, plasma LH concentration did not differ significantly from the control values. The plasma FSH concentration of the oestrogen-treated males showed a delayed rise to values equal to or higher than those of controls of the same age. The delayed rise in plasma FSH concentration in the oestrogen treated males preceded the delayed rise in plasma testosterone in these animals. The decrease in plasma FSH concentration from the high prepubertal values to the lower values in adults occurred at different ages in the control and in oestrogen-treated rats but in both groups the decrease occurred as plasma testosterone levels were increasing and the first wave of spermatogenesis was reaching completion. The increase in plasma FSH concentration after castration was reduced in oestrogen-treated males during the period throughout which FSH levels in the intact animals were subnormal but the levels in oestrogen-treated males castrated after the delayed rise in FSH had occurred did not differ from control values. It is suggested that the delayed sexual maturation of male rats treated with high doses of oestrogen in the neonatal period is related principally to abnormalities in the secretion of FSH.

Effect of 5alpha reduced androgens on sex accessory organs, initiation and maintenance of spermatogenesis in the rat.

The effect of several androgens on initiation and maintenance of spermatogeneSis was studied in newborn estrogen-blocked and in adult hypophysectomized rats. The steroid treatment inhibited testicular and body weight in all animals. Treatment of estrogen-blocked animals with androgens resulted in a stimulation of the growth of sex accessory organs, except in the case of androsterone. Initiation of spermatogenesis and its progression to mid pachytene stage (first wave) was observed in all steroid treated rats. In estrogen-blocked animals the first wave of spermatogenesis proceeded only to pachytene spermatocytes. Administration of testosterone propionate. dihydrotestosterone or 5a-androstanediol to these animals resulted in completion of meiosis and formation of spermatids up to Step 8 of spermiogenesis. Completion of meiosis was not observed in androsterone treated rats. In hypophysectomized animals all 5a-reduced androgens maintained spermatogenesis similar to testosterone.

Effects of gonadotropins and testosterone on the testes of prepubertal mice.

AbstractPituitary follicle‐stimulating hormone FSH supports the synthesis of testicular proteins prior to the onset of spermatid formation. Supplementary FSH enhances the activities of certain enzymes that generate reduced nicotinamide adenine dinucleotide phosphate (NADPH) and induces the division and differentiation of Leydig cell precursors. In addition, exogenous FSH counteracts the effects of continued treatment with testosterone on Leydig cells up to the third week postpartum and offsets the effects of the steroid on the weights of the prepubertal testis and seminal vesicles. In its antagonism of testosterone and its activation of glucose‐6‐phosphate dehydrogenase (G6PDH), FSH clearly differs from the interstitial‐cell‐stimulating hormone ICSH during the first wave of spermatogenesis in the mouse testis.

Effect of the interstitial-cell-stimulating hormone on testicular enzymes during spermatogenesis.

AbstractThe activities of 17β‐hydroxysteroid dehydrogenase and of some NADPH‐generating enzymes in the fetal mouse testis during the formation of the accessory reproductive glands indicate that Leydig cells synthesize androgens before gonadotropins are secreted. In the neonatal testis the seminiferous tubules grow rapidly and the relative volume of the intertubular tissues decreases. At the same time the absolute activity of 17βSDH also decreases but there is no evident loss of enzyme activity as the primary spermatocytes begin meiotic prophase. High levels of NADPH‐generating malic enzyme activity coincide with the onset of meiosis and the development of the spermatids, although the activity of 17βSDH remains constant. Both glucose‐6‐phosphate and isocitrate dehydrogenases, which can be activated by gonadotropin, show strong activity prior to spermiogenesis.Significantly increased activities of 17βSDH and the ME occur in 10‐day postpartum intact mice four hours after a single injection of 0.25 unit ICSH. Exogenous gonadotropin stimulates G6PDH and IDH activities during divisions of spermatogonia five days after birth. The pituitary‐Leydig cell axis is stabilized after the beginning of meiosis so exogenous ICSH has little effect on testicular enzymes during the remainder of the first wave of spermatogenesis. Dependence of Leydig cell activities on pituitary gonadotropin seems to occur during pachytene or the early stages of spermatid formation. Androgen synthesis in the murine testis may be associated with the NADPH‐generating ME and IDH as well as G6PDH which has been implicated in other steroidogenic tissues.