Intermediate Spermatogonia Research Articles

The Spatiotemporal Expression and Localization Implicates a Potential Role for SerpinB11 in the Process of Mouse Spermatogenesis and Apoptosis

In this study, the spatiotemporal expression of SerpinB11 in the mouse testis from postnatal 1–60 d was checked, the SerpinB11 protein strongly localized in the intermediate spermatogonia, B-type spermatogonium, preleptotene spermatocyte, leptonema spermatocyte, zygotene spermatocyte, but weakly localized in the pachytene spermatocyte, diplotene spermatocyte, sphere sperm, and the apoptotic sperm was positive stained of SerpinB11 protein, the localization of cell cycle marker CDK4 and meiosis marker SCP3 were investigated, and the SCP3 and SerpinB11 colocalized in the intermediate spermatogonia, B-type spermatogonium, preleptotene spermatocyte. Taken together, these results suggested that SerpinB11 might involved in spermatogenesis and apoptosis.

Role of the spermatogenic–Sertoli cell interaction through cell adhesion molecule-1 (CADM1) in spermatogenesis

Endocrine and local secretory factors have long been known to be required for spermatogenesis. Evidence has been accumulating in recent years indicating that direct contact between spermatogenic and Sertoli cells is also required for spermatogenesis. Cell adhesion molecules of various types have been found in the mammalian testis that are expressed in spermatogenic and/or Sertoli cells and involved in homophilic and/or heterophilic binding. We have cloned a novel cell adhesion molecule, cell adhesion molecule-1 (CADM1), also known as immunoglobulin superfamily 4A or spermatogenic immunoglobulin superfamily, from the mouse testis. CADM1 belongs to the immunoglobulin superfamily and is composed of three immunoglobulin-like domains, a transmembrane domain, and a short intracellular domain. In the seminiferous epithelium, CADM1 is expressed in intermediate spermatogonia through to early pachytene spermatocytes as well as in elongating spermatids--but not in round spermatids, mature spermatozoa, or Sertoli cells. One of the heterophilic binding partners of CADM1 has proven to be a poliovirus receptor, another member of the immunoglobulin superfamily that is expressed in Sertoli cells. Knockout mice for CADM1 develop male infertility due to defective spermatogenesis. These findings suggest that cell adhesion molecules between spermatogenic and Sertoli cells play essential roles in spermatogenesis.

Desenvolvimento testicular, espermatogênese e concentrações hormonais em touros Angus

Este estudo foi realizado com a finalidade de avaliar a evolução das secreções hormonais e do epitélio seminífero em touros da raça Angus de 10 a 38 semanas de idade. Foram castrados 1 a 5 animais em intervalos de quatro semanas (total de 25 touros) para coleta de amostras do parênquima testicular e do plasma sanguíneo. As variáveis relacionadas ao crescimento testicular, aos aspectos quantitativos da espermatogênese e aos níveis hormonais foram transformadas em logaritmo e avaliadas por meio de análise de variância. O diâmetro dos testículos e túbulos seminíferos e o peso testicular apresentaram variações mais acentuadas após 26 semanas de idade. A porcentagem do parênquima testicular ocupado pelos túbulos seminíferos aumentou de 49,3 para 75,2% durante o experimento. A maioria dos túbulos (>90%) apresentou-se com células de Sertoli somente entre 10 e 14 semanas, mas na 18ª (13,8±1,7%) e 22ª semanas (19±1%), o número de túbulos com gonócitos e espermatogônias aumentou em relação às semanas iniciais. Espermatogônias intermediárias e B predominaram na 26ª semana (24,5±8,2%) e a porcentagem de túbulos com espermatócitos foi mais elevada na 30ª semana (42,3±9,9%). Espermátides arredondadas foram detectadas partir da 26ª semana e, na 38ª semana, 62,3±1,5% dos túbulos seminíferos continham espermátides alongadas ou maduras. As variações mais acentuadas no crescimento testicular e, principalmente, no peso testicular após as 26 semanas coincidiram com o estabelecimento da meiose, com as alterações morfológicas do núcleo e nucléolo das células de Sertoli (indicativos do processo de diferenciação das mesmas), com os níveis reduzidos de androstenediona e os incrementos significativos de testosterona e estradiol 17beta. As associações entre o crescimento testicular e os níveis de FSH e LH na circulação periférica foram menos evidentes.

Expression of Col1a1, Col1a2 and procollagen I in germ cells of immature and adult mouse testis

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. The in vivo procollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies in in vitro culture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis.

Characterization of histone H2A.X expression in testis and specific labeling of germ cells at the commitment stage of meiosis with histone H2A.X promoter-enhanced green fluorescent protein transgene.

To study the complex molecular mechanisms of mammalian spermatogenesis, it would be useful to be able to isolate cells at each stage of differentiation, especially at the stage in which the cells switch from mitosis to meiosis. Currently, no useful marker proteins or gene promoters specific to this important stage are known. We report here a transgenic mouse line that under the control of the promoter for a histone variant, H2A.X, expressed an enhanced green fluorescent protein (EGFP) in cells at the stage of the mitosis-meiosis switch. Endogenous H2A.X is expressed in type A spermatogonia through meiotic prophase spermatocytes in testis and in some somatic cells. However, despite the fact that its expression was driven by the H2A.X promoter, the EGFP expressed in the transgenic mice specifically labeled only the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in transgenic mice containing the -600-base pair H2A.X promoter/EGFP construct. Type A spermatogonia and somatic cells of other organs were not labeled. This expression pattern made it possible to isolate living cells from the testis of the transgenic mice at the stage of the mitosis-meiosis switch in spermatogenesis using EGFP fluorescence.

Expression and functional characterization of the adhesion molecule spermatogenic immunoglobulin superfamily in the mouse testis.

Spermatogenic immunoglobulin superfamily (SgIGSF) is a mouse protein belonging to the immunoglobulin superfamily expressed in the spermatogenic cells of seminiferous tubules. We produced a specific polyclonal antibody against SgIGSF. Western blot analysis of the testes from postnatal developing mice using this antibody demonstrated multiple immunopositive bands of 80-130 kDa, which increased in number and size with the postnatal age. Enzymatic N-glycolysis caused reduction in the size of these bands to 70 kDa, indicating that SgIGSF is a glycoprotein and its glycosylation pattern and extent are developmentally regulated. Immunohistochemical analysis of the adult testis demonstrated that SgIGSF was present in the spermatogenic cells in the earlier steps of spermatogenesis and increased in amount from intermediate spermatogonia through zygotene spermatocytes but was diminished in the steps from early pachytene spermatocytes through round spermatids. After meiosis, SgIGSF reappeared in step 7 spermatids and was present in the elongating spermatids until spermiation. The immunoreactivity was localized primarily on the cell membrane. Consistent with the findings in adult testes, the analysis of the developing testes revealed that SgIGSF was expressed separately in the spermatogenic cells in earlier and later phases. Sertoli cells had no expression of SgIGSF, whereas both SgIGSF immunoprecipitated from the testis lysate and produced in COS-7 cells was shown to bind to the surface of Sertoli cells in primary culture. These results suggested that SgIGSF on the surface of spermatogenic cells binds to some membrane molecules on Sertoli cells in a heterophilic manner and thereby may play diverse roles in the spermatogenesis.

Deleted in azoospermia associated protein 1 shuttles between nucleus and cytoplasm during normal germ cell maturation.

DAZAP1 (Deleted in Azoospermia Associated Protein 1) was originally identified through its interaction with a putative male azoospermia factor, DAZ (Deleted in Azoospermia). It contains 2 RNA-binding domains (RBDs) and a proline-rich C-terminal portion and is expressed most abundantly in testes. We used RNA in situ hybridization and immunocytochemistry to study the expression of Dazap1 in mouse testes. Dazap1 messenger RNA (mRNA) was present predominantly in immature germ cells, between the intermediate spermatogonia and preleptotene spermatocyte stages. The DAZAP1 protein was more abundant in germ cells of later stages of development and showed a dynamic subcellular distribution. High expression of DAZAP1 was first detected in midpachytene spermatocytes in stage VII tubules. In these cells, DAZAP1 was present in both the cytoplasm and the nuclei and was clearly excluded from the sex vesicles. In round spermatids, DAZAP1 was localized mainly in the nuclei, whereas in elongated spermatids, it redistributed to the cytoplasm. The subcellular distribution of DAZAP1 suggests that it shuttles between the nucleus and the cytoplasm and may play a role in mRNA transport and/or localization.

Doxorubicin induces male germ cell apoptosis in rats.

To clarify whether apoptosis is involved in doxorubicin (DXR)-induced testicular toxicity and to identify the target germ cell type, adult Sprague-Dawley rats were treated with a single intravenous dose of DXR (8 or 12 mg/kg) and euthanized at 3, 6, 12, 24, and 48 h subsequently. Histologically, germ cell degeneration was first found 6 h after dosing in meiotically dividing spermatocytes and early round spermatids of seminiferous tubules at stage 1, and subsequently observed in spermatogonia at stages I-VI showing ultrastructural characteristics of apoptosis. Coincident with the appearance of morphological changes, degenerating germ cells were shown to be undergoing apoptosis as revealed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The frequency of TUNEL-labeled germ cells increased in a stage- and cell type-specific manner, the peak of frequency gradually progressing from stage I of seminiferous tubules to later stages with time after dosing, suggesting that the damaged germ cells, especially spermatogonia, gradually underwent the processes leading to apoptosis. DNA laddering on gel electrophoresis was apparent 24 and 48 h after dosing. The results demonstrate that apoptosis plays an important role in the induction of testicular toxicity caused by DXR with meiotically dividing spermatocytes and type A and intermediate spermatogonia as highly vulnerable target cells.

Hormonal regulation of spermatogenesis in the hypophysectomized rat: cell viability after hormonal replacement in adults after intermediate periods of hypophysectomy.

A quantitative analysis of germ-cell populations in normal, hypophysectomized (Hx), and Hx-hormone-treated animals was undertaken during periods of regression that were characterized as intermediate, between short-term and long-term regression of the testis. Twenty-one groups of adult rats were administered either follicle-stimulating hormone (FSH), growth hormone, thyroid-stimulating hormone (TSH), or testosterone (T) in various doses and combinations. The dosage of T administered was less than that expected to achieve maximum testis weight. Flutamide and Casodex were used to compete with androgen binding to receptors in Hx animals, as it is known that small amounts of androgen are secreted in the absence of pituitary stimulation. Follicle-stimulating hormone, T, and TSH all significantly maintained testis weight as compared with Hx controls, although FSH and T, singly or in combination, were the most effective. Contamination of the TSH preparation with trace amounts of FSH was apparently responsible for the slight maintenance of testis weight. A novel assay for determination of the numbers of viable germ cells was used in a subset of these groups to determine the cellular sites of FSH and T action. Numbers of type A spermatogonia were lowered after Hx and were maintained by either FSH or T or a combination of these hormones. Other phases of germ-cell development most susceptible to FSH and/or T were the successive conversions of type A spermatogonia to intermediate spermatogonia, intermediate spermatogonia to type B spermatogonia, preleptotene spermatocytes to pachytene spermatocytes, and early pachytene spermatocytes to intermediate maturity pachytene spermatocytes during early and midcycle phases of pachytene spermatocyte development. Germ-cell loss during meiosis and virtually every phase of spermatid development was largely prevented by FSH or T or a combination of these hormones. Thus, in testes in advanced stages of regression, both FSH and T were capable of preventing cell loss, suggesting that both hormones can affect the survival of the same cell type. The present study demonstrated that FSH can partially compensate for lowered T levels. The combined administration of FSH and T was more effective in preventing overall cell degeneration than either hormone alone. Unlike the initial phase of spermatogenesis, in which there is a largely midcycle loss of germ cells due to Hx, the loss of cells during testis regression is more widespread and impacts several cell types in more than one stage of the spermatogenic cycle.

Hormonal regulation of spermatogenesis in the hypophysectomized rat: FSH maintenance of cellular viability during pubertal spermatogenesis.

The potential for follicle-stimulating hormone (FSH) to promote germ-cell survival and the cellular sites of FSH action were studied using a gonadally maturing (pubertal), hypophysectomized (Hx) rat model in which residual testosterone (T) activity was blocked by injections of an androgen-receptor antagonist, flutamide. Recombinant human FSH was given to androgen-deprived and androgen-blocked male rats at 27 days of age to determine maintenance of individual germ-cell types at 35 days of age. Follicle-stimulating hormone significantly increased testis weights and tubular diameters as compared with Hx and Hx-flutamide controls, although testis weights in FSH-treated animals were significantly lower than in pituitary-intact animals. Morphometric assays to determine ratios of germ cells to Sertoli cells and to determine the number of germ cells present per hour of development showed that the population of type A spermatogonia in the early stages of the cycle was not responsive to FSH. Follicle-stimulating hormone had a marked ability to maintain cell viability in the rapid, successive divisions that begin in the latter part of the cycle and that continue through the next cycle (i.e., from type A1 to A4 and from intermediate spermatogonia to type B spermatogonia to preleptotene spermatocytes to leptotene/zygotene spermatocytes to young pachytene spermatocytes). The data also suggest T responsiveness of these cell types since the Hx-FSH-flutamide group showed lower cell viability at the aforementioned steps when compared with the Hx-FSH group. Too few cell types were present at subsequent phases of spermatogenesis to allow a sensitive determination of FSH activity in the maintenance of cell viability. The data show the potential of FSH in the absence or relative absence of T activity to maintain cell viability. These data support the concept of overlapping and synergistic (or additive) effects of T and FSH in the immature rat and identify the cellular sites of FSH action.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Alteration of mRNA transcript levels of rat testicular cells following procarbazine administration.

The present study examined the effects of a single 400-mg/kg dose of procarbazine upon various testicular mRNA transcript levels in adult Sprague-Dawley rats. Northern blot hybridization was employed to measure the steady-state mRNA levels of proteins specific for Sertoli cells (adrogen-binding protein [ABP] and transferrin) and spermatids (protamine 1 and transitional protein 2). In addition, mRNA transcript levels were determined for germ cell-specific hemiferrin and insulin-like growth factor-1 (IGF-1), which in adult rats is present predominantly in primary spermatocytes. Furthermore, the chronology of the effects of procarbazine upon spermatogonial populations was examined in whole mounts of seminiferous tubules. The effect of procarbazine upon spermatogenesis was first noted among mature A3, young A4, and B spermatogonia 48-72 hours after drug administration. These results indicate that A2 through intermediate spermatogonia were most susceptible to procarbazine. In addition, degeneration of spermatocytes and young spermatids was evident by 5 days. Northern blot analysis of testicular poly (A)+ RNA revealed that the steady-state levels of mRNA transcripts of the spermatid nuclear proteins (protamine 1, 700 bp; and transitional protein 2, 580 bp) remained relatively unchanged for 5 days after procarbazine administration but were significantly decreased by more than 70% by 7 days. On the other hand, the steady-state mRNA levels for the Sertoli cell proteins ABP (1.7 kb) and transferrin (2.7 kb) were decreased by 45% and 50% (P < 0.05), respectively, 2 days after procarbazine administration. Significant suppression of ABP mRNA levels persisted through day 5, with partial recovery noted on day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of prenatal and postnatal photoperiod on spermatogenic development in the Djungarian hamster (Phodopus sungorus sungorus).

The effect of the pre- and postnatal daylength on the start of spermatogenesis and further testicular development from day 4 up to day 127 was investigated in Djungarian hamsters. Hamsters were either gestated under long (16 h light:8 h dark) photoperiod and reared under long or short (4 h light:20 h dark) photoperiod after birth (L/L and L/S hamsters, respectively), or gestated under short photoperiod and transferred to long photoperiod after birth (S/L hamsters). In L/L and L/S hamsters, spermatogenesis started between day 4 and day 5 (day of birth = day 1), when the first gonocytes entered the S-phase. A, Intermediate and B spermatogonia were first observed on days 6, 8 and 9, respectively. The proliferation pattern of gonocytes and Sertoli cells, studied between day 4 and day 9, did not differ between L/L and L/S hamsters. Hence, the duration of the postnatal photoperiod had no effect on the start of spermatogenesis. The first effect of postnatal photoperiod on spermatogenic development was observed on day 15, when testis weights and tubular diameters were reduced in L/S animals. From day 22 onwards, spermatogenesis was arrested mainly at the mid-pachytene stage, no tubular lumen was formed, and the number of preleptotene spermatocytes was reduced. The ultimate number of Sertoli cells per testis was not affected by postnatal short photoperiod. The duration of the prenatal photoperiod had a clear effect on spermatogenesis after birth. In S/L hamsters, the number of gonocytes per tubular cross-section was reduced on day 4 and 4.5. Gonocyte proliferation was reduced on day 5 and spermatogenesis started one day later. Consequently, A and Intermediate spermatogonia appeared on day 7 and 9, respectively. Sertoli cell proliferation was also shifted to later ages, but the ultimate number of Sertoli cells did not differ from L/L or L/S hamsters. From day 29 onwards, the number of preleptotene spermatocytes was increased in S/L hamsters, indicating that the Sertoli cells in these animals could support more germinal cells. In conclusion, a short postnatal photoperiod does not affect spermatogenesis before day 15 after birth, when further testicular development becomes arrested. A short prenatal photoperiod delays the start of spermatogenesis by one day, alters the proliferation pattern of Sertoli cells, and from day 29 onwards, enables the Sertoli cells to support more germinal cells. The duration of the pre- and postnatal photoperiod did not affect the ultimate number of Sertoli cells.

Proliferation of spermatogonia and Sertoli cells in maturing mice.

Spermatogonial proliferation was studied in mice from day 13 p.p. when the seminiferous epithelium is incomplete, until week 12 p.p. when a steady state at adult levels has been attained. Counts of undifferentiated, A1 and intermediate spermatogonia and primary spermatocytes in stages IV and IX of the cycle of the seminiferous epithelium were made in whole mounted seminiferous tubules. Sertoli cell proliferation was studied in a separate series from 6 to 14 days p.p. employing the 3H-thymidine labeling index. It appeared that 1. Sertoli cell proliferation stops at day 12 whereafter the cells obtain their adult appearance; 2. The numbers of stem cell spermatogonia and the production of differentiating A1 spermatogonia increase almost twofold between day 13 and week 12; 3. The efficiency of the divisions of the differentiating A1-B spermatogonia is similar to that in the adult throughout this period; 4. At all ages studied, the cell counts revealed an almost constant numerical relationship between Sertoli cells and germ cells, which suggests a function of Sertoli cells in the regulation of spermatogonial proliferation.

Spermatogenesis in the immature mouse proceeds faster than in the adult.

The first appearance of spermatogenic cell types related to the age of the animal was studied in sections and tubular whole mounts of testes of normal mice (Cpb-N strain) up to 34 days pp. The first intermediate spermatogonia and leptotene spermatocytes were seen at days 4 and 7 p.p., respectively. It was found that the subsequent types of spermatogenic cells appear earlier than could be expected if spermatogenesis was to proceed at adult speed. [3H]thymidine labelling studies revealed that within a given interval of time, spermatocytes and spermatids in immature mice develop into more advanced cell types than in adults. The labelling studies and the observation that the cellular associations are always identical to those in the adult, indicate that the rate of acceleration in young mice is the same for spermatogonia, spermatocytes and spermatids. The mean duration of the cycle of the seminiferous epithelium during the age interval of 10 to 30 days p.p. is 7.51 +/- 0.10 days, compared to 8.61 +/- 0.08 in the adult. It increases gradually towards the adult level, reaching the value of 8.45 +/- 0.17 days between days 33 and 56 p.p.

The effects of negative pions on spermatogonial survival in the mouse.

A study of the radiation effects of pi-mesons on the testes of the mouse has been carried out using the pion beam at the SRC Rutherford Laboratory. The cell killing effects on type B and intermediate spermatogonia have been assessed and compared with the effects of 20-0 kVp X rays, delivered at the same dose rate. It was found that not only did irradiation of the testes not disturb the kinetics of spermatogenesis, but also the RBE for pions measured in the Bragg peak and on the plateau was not significantly different from unity. The comparison of this result with those of others using other pion beams is discussed.

Timing and localization of the stimulatory effect of follicle-stimulating hormone on uridine incorporation in the mouse testis in vivo.

Human pituitary FSH increased the incorporation of [5-(3)H]uridine into RNA in vivo in the testes of intact 9-day-old mice and of hypophysectomized adults. In both groups the effect was greatest 8 h after administration of 5--7.5 i.u. FSH. Autoradiographs were prepared from the testes of hypophysectomized adult mice given subcutaneous injections of FSH or of 0.9% saline 6 h, and [5-(3)H]uridine 1.5 h, before death. Treatment with FSH caused statistically significant increases in the density of silver grains over the nuclei of Sertoli cells, type A and intermediate spermatogonia, and preleptotene and mid-pachytene primary spermatocytes. It was concluded that FSH has a generalized stimulatory action on RNA synthesis in the nuclei of Sertoli cells and those types of germinal cell which synthesize RNA most actively.

Actions of an antispermatogenic, but non-mutagenic, indenopyridine derivative in mice and Salmonella typhimurium

This paper describes a new antispermatogenic agent. Following single oral administration to mice, the indenopyridine derivative (4aRS, 5SR, 9bRS)-2-ethyl-1,3,4,4a,5, 9b-hexahydro-7-methyl-5- p-tolyl-2 H-indeno(1,2-c) pyridine hydrochloride, code No. 20-438, produced long-lasting inhibition of the spermatogenic process at dose levels of 10 mg/kg ( 1 40 of the lowest lethal dose) and higher. Testes weights were significantly reduced from days 2–217 after treatment, and no clear-cut evidence of a recovery was found during this time. The fertility of treated males was normal during the initial 2 weeks after treatment, followed by partial or total sterility in weeks 3–6, and incomplete recovery in weeks 7–29 after treatment. The antifertility effects were caused by maturation depletion of the germ cells, leading to oligospermia. The following rank of decreasing “susceptibility” of the germ cells was observed: Spermatocytes > early spermatids, intermediate spermatogonia > stem cells. Sperm and late spermatids were not affected. Despite the characteristic specific germ-cell pattern of antifertility effects, 20–438 showed neither indications of pre- and post-implantational dominant lethality, nor mutagenic potentiality as measured by cytogenetic analysis of spermatocytes or spermatogonia, the sperm abnormality assay, the micronucleus test, and the Salmonella assay. These data suggest that the action of 20–438, leading to oligospermia, does not involve genetic toxic effects.

The population of germ cells in immature male rats following irradiation at birth.

Male rats were irradiated with 19 r on the day of birth, and killed at intervals ranging from 5 to 18 days. Estimates were made of the absolute and relative numbers of germ cells at different stages of spermatogenesis in 64 irradiated and 61 untreated specimens. In the normal rat, the calculated population of germ cells increased from about 160000 at 5 days to 30 million at 18 days. Only negligible numbers of primordial germ cells (gonocytes and transitional cells) persisted beyond the age of 10 days. Small numbers of spermatogonia type A appeared at 5 days (15000) and their population rose to about 1 million at 12 days, and 2 million at 18 days (7 % of all germ cells). Intermediate spermatogonia first occurred in appreciable numbers (23000 to 55000) at 8 or 9 days, when the population of type-A spermatogonia was 360000. The subsequent rise in the population of intermediate spermatogonia was more rapid than that of type A (4 million at 18 days). Spermatogonia type B and primary spermatocytes appeared at 9 to 10 days, and their numbers rose more steeply still (6.5 and 16 million at 18 days, respectively). Irradiation at birth exerted no rapid effect on the cytological appearance of primordial germ cells. Transformation from gonocytes to transitional cells appeared to proceed normally and the estimated total population of germ cells at 5 days was no smaller than in the controls. Subsequently, however, many of the transitional cells failed to divide: they enlarged to form giant cells, acquired bizarre nuclear outlines, and persisted for unusually long periods. Some degenerated at mitotic prophase or metaphase, while a few seemed to die at interphase, without entering division. The calculated total population of germ cells in irradiated rats rose from 160000 at 5 days to 9.4 million at 18 days. Small numbers of spermatogonia type A, presumably derived from such primordial germ cells as were able to complete mitosis, appeared some 2 to 3 days later than in controls. The number of type-A spermatogonia in 7-day-old irradiated rats was 44000, cf. 215000 in controls; the difference became less pronounced with time, and by the age of 18 days, the population of 1.9 million was comparable to that estimated for the controls. Small numbers of intermediate spermatogonia appeared on the 9th (8000) and 10th day (35000), when the population of type-A spermatogonia was about 110000 and 260000 respectively. By the 18th day, intermediate spermatogonia numbered 2 million. The populations of type-B spermatogonia and primary spermatocytes rose from 11000 to 13000 at 10 days to 1.6 and 3.4 million, respectively, at 18 days. The difference in the absolute and relative numbers of germ cells between normal and irradiated testes widened progressively with advance in the developmental stage of the germ cells. Analysis of the results indicates that in the reduced population of spermatogonia type A after irradiation, the pattern of spermatogonial mitoses is modified so as to favour the formation of more type-A, in preference to intermediate, spermatogonia.

DURATION OF SPERMATOGENESIS AND SPERMATOZOAN TRANSPORT IN THE RABBIT BASED ON CYTOLOGICAL CHANGES, DNA SYNTHESIS AND LABELING WITH TRITIATED THYMIDINE.

AbstractThe duration of spermatogenesis in the rabbit was determined by injecting in a single dose 1 millicurie of thymidine‐methyl‐H3 per kg into the marginal ear vein of each of 14 Dutch‐belted bucks. At different intervals, biopsies and unilateral castrations were performed on nine of the 14 bucks to time the stages of spermatozoan formation in the testis and transport through the epididymis. The remaining five animals were ejaculated for 63 days to determine the interval between the thymidine‐H3 injection and the appearance of the isotope in the ejaculated semen.Histological sections, 8 μ thick, were stained with the Feulgen technique and coated with NTB 3 nuclear emulsion. The autoradiographs showed that the leptotene primary spermatocytes were the most advanced spermatogenic cells that incorporated the label. This agreed with microspectrophotometric determinations of DNA which showed this to be the last stage at which DNA was synthesized in spermatogenesis. Therefore there appears to be no DNA exchanged during spermiogenesis. Uptake of tritiated thymidine by spermatogonia also was in accordance with measurements of DNA synthesis and content of these cells.Thirty‐one days post‐injection the label had progressed from leptotene primary spermatocytes to spermatozoa leaving the testis. At this time a few labeled spermatozoa also were present in semen smears taken from the caput epididymis. Epididymal transport time was eight days for one rabbit and ten days for four rabbits. The duration of one cycle of the seminiferous epithelium based on 55 estimates was 10.9 ± 0.1 days. The lifespan of type A spermatogonia, type 1 intermediate spermatogonia, type 2 intermediate spermatogonia, type B spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa was estimated to be 3.4, 0.6, 2.8, 1.1, 16.5, 0.8, 10.0 and 15.6 days respectively.The estimated total duration of spermatogenesis in the rabbit depends on the point chosen as the onset of spermatogenesis. If spermatogenesis is considered to begin with the first of the series of spermatogonial divisions leading to the production of primary spermatocytes then about four cycles of the seminiferous epithelium or 4 × 10.9 = 43.6 days are required. However, if one assumes that spermatogenesis starts with the formation of spermatogonial stem cells and that the lifespan of these stem cells is one cycle of the seminiferous epithelium then spermatogenesis extends over about 4.75 cycles, or 51.8 days.