Gonadotrophin-releasing hormone antagonist arrests premeiotic germ cell proliferation but does not inhibit meiosis in the male monkey: a quantitative analysis using 5-bromodeoxyuridine and dual parameter flow cytometry

Meiosis constitutes a crucial phase of spermatogenesis since the recombination of genetic information and production of haploid round spermatids need to be achieved. Although it is well established that gonadotrophic hormones are required for completion of the spermatogenic process, little is known about the dynamic and kinetic aspects of development of spermatocytes into spermatids and its endocrine control in the primate. In this study, S-phase germ cells were labelled using 5-bromodeoxyuridine (BrdU) incorporation and were then followed throughout meiosis under normal conditions and following GnRH antagonist (ANT)-induced gonadotrophin withdrawal in a nonhuman primate model, the cynomolgus monkey (Macaca fascicularis). Adult animals received either vehicle (VEH, n = 4) or the ANT cetrorelix (n = 5) throughout 25 days. On day 7 all animals received a bolus injection of BrdU. A biopsy was performed after 3 h, one testis was removed 9 days later (day 16 of treatment) and the other testis after 18 days (day 25 of treatment). Serum testosterone and inhibin levels, and testis weight were reduced (P < 0.05) by ANT treatment. BrdU localized to pachytene spermatocytes 9 days after BrdU and to round spermatids 18 days after BrdU in both groups, demonstrating that BrdU-labelled pachytene spermatocytes had undergone meiosis. Flow cytometric analysis revealed that the relative number and number per testis of BrdU-tagged 2C and 4C cells were reduced significantly (P < 0.05) within 16 days of ANT treatment. Numbers of 1C cells were lowered by day 25. The cell ratio for 1C:4C was similar with VEH and ANT (P > 0.05). These findings indicate that ANT reduced the number of cells available for meiosis but did not alter the rate of transition into round spermatids. Unexpectedly, however, the stage-dependent progression of BrdU-tagged round spermatids was significantly (P < 0.05) retarded under ANT as seen from the frequency of tubules containing BrdU-labelled round spermatids. The average duration of spermatogenic cycle was slightly prolonged (9.8 days in the VEH group and 10.8 days in the ANT group (P = 0.09)). Since no atypical germ cell associations could be found, it remains unclear whether this slight prolongation is entirely due to altered spermatid progression or whether earlier phases are affected. We conclude for the nonhuman primate that (1) BrdU-labelling of premeiotic germ cells is suitable for tracing their meiotic transition into postmeiotic cells, (2) unlike in the rat, gonadotrophin suppression initially affects premeiotic cell proliferation and thus the number of cells available for meiosis, (3) the meiotic process continues quantitatively despite gonadotrophin deficiency and (4) prolonged gonadotrophin deficiency might alter the timing of germ cell development.

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Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽

10.1210/en.2007-1535 ◽

2007 ◽

Vol 149 (4) ◽

pp. 1813-1819 ◽

Cited By ~ 47

Author(s):

Eri Shiraishi ◽

Norifumi Yoshinaga ◽

Takeshi Miura ◽

Hayato Yokoi ◽

Yuko Wakamatsu ◽

...

Keyword(s):

Cell Proliferation ◽

Germ Cell ◽

Germ Cells ◽

Sex Differentiation ◽

Somatic Cells ◽

Oryzias Latipes ◽

Cell Number ◽

Gonadal Differentiation ◽

Loss Of Function ◽

Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

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Germ cell fate and seminiferous tubule development in bovine testis xenografts

Reproduction ◽

10.1530/rep.1.00912 ◽

2005 ◽

Vol 130 (6) ◽

pp. 923-929 ◽

Cited By ~ 54

Author(s):

Rahul Rathi ◽

Ali Honaramooz ◽

Wenxian Zeng ◽

Stefan Schlatt ◽

Ina Dobrinski

Keyword(s):

Germ Cell ◽

Cell Fate ◽

Germ Cells ◽

Cell Number ◽

Sperm Production ◽

Continuous Increase ◽

Control Tissue ◽

Low Efficiency ◽

Pachytene Spermatocytes ◽

Testis Tissue

Spermatogenesis can occur in testis tissue from immature bulls ectopically grafted into mouse hosts; however, efficiency of sperm production is lower than in other donor species. To elucidate a possible mechanism for the impaired spermatogenesis in bovine testis xenografts, germ cell fate and xenograft development were investigated at different time points and compared with testis tissue from age-matched calves as controls. Histologically, an initial decrease in germ cell number was noticed in xenografts recovered up to 2 months post-grafting without an increase in germ cell apoptosis. From 2 months onward, the number of germ cells increased. In contrast, a continuous increase in germ cell number was seen in control tissue. Pachytene spermatocytes were observed in some grafts before 4 months, whereas in the control tissue they were not present until 5 months of age. Beyond 4 months post-grafting spermatogenesis appeared to be arrested at the pachytene spermatocyte stage in most grafts. Elongated spermatids were observed between 6 and 8 months post-grafting, similar to the controls, albeit in much lower numbers. Lumen formation started earlier in grafts compared with controls and by 6 months post-grafting tubules with extensively dilated lumen were observed. A donor effect on efficiency of spermatogenesis was also observed. These results indicate that the low efficiency of sperm production in bovine xenografts is due to an initial deficit of germ cells and impaired meiotic and post-meiotic differentiation. The characterization of spermatogenic efficiency will provide the basis to understand the control of spermatogenesis in testis grafts.

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Hepatocyte growth factor modulates in vitro survival and proliferation of germ cells during postnatal testis development

Journal of Endocrinology ◽

10.1677/joe.1.06528 ◽

2006 ◽

Vol 189 (1) ◽

pp. 137-146 ◽

Cited By ~ 11

Author(s):

A Catizone ◽

G Ricci ◽

J Del Bravo ◽

M Galdieri

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Germ Cell ◽

Germ Cells ◽

Cell Types ◽

Tyrosine Kinase Receptor ◽

Testis Development ◽

Pachytene Spermatocytes ◽

Hepatocyte Growth

The hepatocyte growth factor (HGF) is a pleiotropic cytokine that influences mitogenesis, motility and differentiation of many different cell types by its tyrosine kinase receptor c-Met. We previously demonstrated that the c-Met/HGF system is present and functionally active during postnatal testis development. We found also that spermatozoa express c-Met and that HGF has a positive effect on the maintenance of sperm motility. In the present paper, we extend our study on the germ cells at different stages of differentiation during the postnatal development of the testis. We demonstrate that c-met is present in rat spermatogonia, pachytene spermatocytes and round spermatids and that HGF significantly increases spermatogonial proliferation in 8- to 10-day-old pre-pubertal rats. At this age HGF does not affect Sertoli cells and peritubular myoid cells proliferation. In addition, we studied the effect of the factor on germ cell apoptosis and we show that HGF prevents the germ cell apoptotic process. We also studied the effect of HGF on 18- to 20-day-old and 28- to 30-day-old rat testes. At these ages also the factor significantly increases germ cell duplication and decreases the number of apoptotic cells. However, the effect on programmed cell death is higher in the 8- to 10-day-old rats and declines in the older animals. In conclusion, we report that rat germ cells (spermatogonia, pachytene spermatocytes and round spermatids) express c-met and that HGF modulates germ cell proliferating activity and apoptosis in vitro. These data indicate that the c-Met/HGF system is involved in male germ cell homeostasis and, consequently, has a role in male fertility.

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236. Effects of long term recombinant rat follicle-stimulating hormone replacement on the restoration of spermatogenesis after chronic suppression of gonadotrophins in adult rats

Reproduction Fertility and Development ◽

10.1071/srb08abs236 ◽

2008 ◽

Vol 20 (9) ◽

pp. 36

Author(s):

S. M. Ruwanpura ◽

P. G. Stanton ◽

D. M. Robertson ◽

R. I. McLachlan ◽

Y. Makanji ◽

...

Keyword(s):

Sertoli Cell ◽

Germ Cells ◽

Follicle Stimulating Hormone ◽

Inhibin B ◽

Testis Weight ◽

Adult Rats ◽

Early Pachytene ◽

Pachytene Spermatocytes ◽

Stimulating Hormone

Follicle stimulating hormone (FSH) in short-term rat studies supports spermatogenesis at multiple levels, notably spermatogonial development. The role of FSH in supporting full spermatogenesis in rats is still in question as long-term studies have not been possible due the development of neutralising antibodies to heterologous FSH preparations. This study sought to assess the effects of a homologous recombinant rat FSH (rr-FSH) preparation on the long-term restoration of spermatogenesis. Adult rats were GnRH-immunised (GnRH-im) for 12 weeks then, administered an anti-androgen; flutamide (flut), alone or together with rr-FSH (8µg/rat/daily) for 56 days (1 spermatogenic cycle). Germ and Sertoli cell numbers were quantified using an optical disector stereological method. Testis weight, serum FSH and inhibin B and Sertoli cell nuclear volume were significantly reduced to 15%, 13%, 25% and 57% of controls respectively, following GnRH-im+flut treatment. GnRH-im+flut treatment reduced A/I spermatogonial, type B spermatogonial+preleptotene, leptotene+zygotene and early pachytene spermatocyte numbers to 28%, 68%, 50% and 19% (P < 0.001) of controls respectively, with later germ cells rarely observed. After FSH treatment, no significant affect on testis weight, serum FSH and inhibin B or Sertoli cell number were observed. However, rr-FSH treatment significantly increased numbers of A/I spermatogonia, leptotene+zygotene and early pachytene spermatocytes from 28 = >42%, 50 = >69% and 19 = >27% of controls, respectively, while no differences were observed in later germ cell types. rr-FSH also increased (P < 0.05) the volume of Sertoli cell nuclei from 57 = >66% of control. In conclusion, FSH is unable to support full rat spermatogenesis; however, FSH can partially support germ cells notably spermatogonia through to early pachytene spermatocytes, despite the absence of androgenic support.

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Evaluation of the role of germ cells in regulating the route of secretion of immunoactive inhibin from the rat testis

Journal of Endocrinology ◽

10.1677/joe.0.1320439 ◽

1992 ◽

Vol 132 (3) ◽

pp. 439-NP ◽

Cited By ~ 7

Author(s):

S. Maddocks ◽

J. B. Kerr ◽

G. Allenby ◽

R. M. Sharpe

Keyword(s):

Germ Cell ◽

Sertoli Cell ◽

Germ Cells ◽

Cell Types ◽

Cell Depletion ◽

Normal Adult ◽

Adult Testis ◽

Pachytene Spermatocytes ◽

Blood Testis Barrier

ABSTRACT During normal sexual maturation of the male rat there is a progressive change in the route of secretion of inhibin by the Sertoli cell, from a predominantly basal route of secretion in prepuberty to a predominantly apical route of secretion in adulthood. This change may be monitored by comparing the levels of inhibin in testicular (TV), spermatic and peripheral (PV) venous blood and the levels in testicular interstitial fluid (IF). This study has assessed the role of germ cells in effecting this change by assessing (a) the effect of total germ cell depletion by X-irradiation of the males in utero, and (b) the effect of selective germ cell depletion in adulthood using the testicular toxicant, methoxyacetic acid (MAA). Female rats were X-irradiated on day 20 of gestation to produce male offspring whose testes were germ-cell deficient. Blood and IF samples were collected from groups of these offspring and age-matched controls at 35 and 100 days of age. In blood and IF samples, inhibin concentrations were significantly higher at 35 days of age than at 100 days. The absence of germ cells in X-irradiated animals did not affect the age-related fall in inhibin levels, nor the change in the predominant route of secretion of inhibin from the testis into blood. Testosterone was almost undetectable in 35-day-old controls, but was raised significantly by 100 days of age. In X-irradiated animals, testosterone levels were increased significantly at 35 days of age, and the levels in most samples were increased even more substantially by 100 days of age. However, PV levels of testosterone in 100-day-old X-irradiated animals were significantly lower than in controls. LH and FSH levels were raised in X-irradiated animals compared with their age-matched controls, but FSH levels in X-irradiated animals still fell with age, as in the controls. The role of specific germ cell types in regulating the route of secretion of inhibin from the normal adult testis was studied after depletion (80–100%) of pachytene and later spermatocytes by a single oral administration of MAA (650 mg/kg) to adult rats. At 3 days after MAA treatment, coincident with the loss of pachytene spermatocytes, plasma inhibin levels were increased significantly in blood and IF samples, and this was associated with a dramatic change in the route of secretion of inhibin from the testis, with increased secretion of this peptide via the base of the Sertoli cell into IF and TV blood. However, previous studies suggest that this may be a consequence of direct stimulation by MAA, rather than the absence of pachytene spermatocytes. By 21 days after MAA treatment, when late-stage spermatids are absent, plasma inhibin levels were reduced significantly compared with controls, although the route of secretion of inhibin from the testis was comparable with that of controls. By 42 days, when a normal germ cell complement has been restored, plasma concentrations and the route of secretion of inhibin from the testis were similar to controls. It is concluded that: (1) the presence of germ cells is not necessary for the maturational changes in the rate and route of secretion of inhibin by the Sertoli cell; these changes are most likely a consequence of formation of the blood–testis barrier, (2) in the normal adult testis, MAA-induced depletion of the most mature germ cell types affects the rate, but not the route, of inhibin secretion, whilst depletion of pachytene spermatocytes affects both parameters; the latter may indicate an early effect of MAA on the functional competence of the blood–testis barrier. Journal of Endocrinology (1992) 132, 439–448

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Effects of subchronic exposure to carbendazim on spermatogenesis and fertility in male rats

Toxicology and Industrial Health ◽

10.1177/0748233709103033 ◽

2009 ◽

Vol 25 (1) ◽

pp. 41-47 ◽

Cited By ~ 24

Author(s):

G Yu ◽

Q Guo ◽

L Xie ◽

Y Liu ◽

X Wang

Keyword(s):

Germ Cells ◽

Flow Cytometric Analysis ◽

Testicular Tissue ◽

Male Rats ◽

Seminiferous Tubules ◽

Control Group ◽

Testis Weight ◽

Subchronic Exposure ◽

Flow Cytometric ◽

Sperm Counts

The aim of this study is to investigate the effects of subchronic exposure to carbendazim on spermatogenesis and fertility in male rats. Ninety-eight healthy male rats were divided into four groups: three exposure groups and a control group. Carbendazim was administered orally to male rats at 0, 20, 100 and 200 mg/kg for 80 days prior to mating. Each male was cohabited with an unexposed female for a maximum of 5 days. In 100 and 200 mg/kg groups, the mating index was relatively increased, the fertility index was decreased, and the testis weight, the sperm counts and motility were also decreased. The levels of luteinizing hormone (LH) showed a decreasing tendency and there was a statistical difference between the 200 mg/kg group and the control group. There were no obvious effects on the levels of follicle stimulating hormone (FSH) and testosterone (T). Histopathological evaluation showed atrophic seminiferous tubules, decreased germ cells, and increased sloughing of germ cells. Flow cytometric analysis of the testicular tissue revealed that carbendazim inhibited meiotic transformation and interfered with the spermatogenic process. These results suggest that carbendazim has adverse effects on spermatogenesis, resulting in reduced fertility in male rats.

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Expression of Bcl-2 and 3-ß hydroxysteroid dehydrogenase protein during oocyte and follicle development in fetal and post-natal pig ovaries

Reproduction Fertility and Development ◽

10.1071/rd00003 ◽

1999 ◽

Vol 11 (8) ◽

pp. 463 ◽

Cited By ~ 12

Author(s):

Wesley M. Garrett ◽

H. David Guthrie

Keyword(s):

Cell Proliferation ◽

Germ Cell ◽

Germ Cells ◽

Granulosa Cells ◽

Stromal Cells ◽

Post Partum ◽

Nuclear Antigen ◽

Ki 67 ◽

Primordial Follicles ◽

Small Clusters

The fetal and post-natal development of the pig ovary involves both proliferation and apoptotic loss of germ cells, follicle formation and growth, and the initiation of oocyte meiotic maturation. The present study measured the expression of the proto-oncogene Bcl-2 immunohistochemically on paraffin sections of pig ovaries to determine its relationship with folliculogenesis on Days 50 and 80 post coitum (p.c.) and on Days 1, 21, and 56 post partum (p.p.). The expression of the steroidogenic enzyme 3β-hydroxy-steroid dehydrogenase (3βHSD) was used to determine the lineages of the cells forming the ovarian follicles, and the expression of the cell proliferation-associated nuclear antigen Ki-67 was used to determine germ cell proliferation and the initiation of follicle growth. Expression of Ki-67 showed that many oogonia were proliferating on Days 50 and 80 p.c. Granulosa cells were more proliferative on Day 56 p.p. than at any other stage; Ki-67 was expressed in 70% of growing follicles and granulosa cells had a 3% mean staining index per section. Less than 4% of germ cells and follicles had morphological signs of degeneration during the period of the study. Bcl-2 was most abundant on Days 21 p.p. and 56 p.p.; staining was localized to stromal cells among follicles and in small clusters in the cortical–medullary junction (CMJ). 3βHSD staining on Day 50 p.c. was seen in cords of stromal cells within the medulla of the ovary, and in the stromal cells investing the oogonial nests. On Days 80 p.c., 1 p.p., 21 p.p., and 56 p.p., 3βHSD was expressed in the granulosa cells of primary or primordial follicles at the CMJ. Production of Bcl-2 by somatic cells may support germ cell and preantral follicle survival.

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VEGF/VEGFR2 Signaling Regulates Germ Cell Proliferation in vitro and Promotes Mouse Testicular Regeneration in vivo

Cells Tissues Organs ◽

10.1159/000440949 ◽

2016 ◽

Vol 201 (1) ◽

pp. 1-13 ◽

Cited By ~ 12

Author(s):

Ruhui Tian ◽

Shi Yang ◽

Yong Zhu ◽

Shasha Zou ◽

Peng Li ◽

...

Keyword(s):

Cell Proliferation ◽

Germ Cell ◽

Germ Cells ◽

Seminiferous Tubules ◽

Testicular Development ◽

Male Germ Cells ◽

Vegfr2 Signaling ◽

Proliferation In Vitro ◽

Germ Cell Proliferation

Vascular endothelial growth factor (VEGF) plays fundamental roles in testicular development; however, its function on testicular regeneration remains unknown. The objective of this study was to explore the roles VEGF/VEGFR2 signaling plays in mouse germ cells and in mouse testicular regeneration. VEGF and the VEGFR2 antagonist SU5416 were added to culture medium to evaluate their effects on spermatogonial stem cell line (C18-4 cells) proliferation. Testicular cells obtained from newborn male ICR mice were grafted into the dorsal region of male BALB/c nude mice. VEGF and SU5416 were injected into the graft sites to assess the effects of the VEGF and VEGFR2 signaling pathways on testicular reconstitution. The grafts were analyzed after 8 weeks. We found that VEGF promoted C18-4 proliferation in vitro, indicating its role in germ cell survival. HE staining revealed that seminiferous tubules were reconstituted and male germ cells from spermatogonia to spermatids could be observed in testis-like tissues 8 weeks after grafting. A few advantaged male germ cells, including spermatocytes and spermatids, were found in SU5416-treated grafts. Moreover, VEGF enhanced the expression of genes specific for male germ cells and vascularization in 8-week grafts, whereas SU5416 decreased the expression of these genes. SU5416-treated grafts had a lower expression of MVH and CD31, indicating that blockade of VEGF/VEGFR2 signaling reduces the efficiency of seminiferous tubule reconstitution. Collectively, these data suggest that VEGF/VEGFR2 signaling regulates germ cell proliferation and promotes testicular regeneration via direct action on germ cells and the enhancement of vascularization.

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Molecular mechanism of green microalgae, Dunaliella salina, involved in attenuating balloon injury-induced neointimal formation

British Journal Of Nutrition ◽

10.1017/s0007114510000693 ◽

2010 ◽

Vol 104 (3) ◽

pp. 326-335 ◽

Cited By ~ 4

Author(s):

Ming-Jyh Sheu ◽

Hsu-Chen Cheng ◽

Yi-Chung Chien ◽

Pei-Yu Chou ◽

Guang-Jhong Huang ◽

...

Keyword(s):

Cell Proliferation ◽

Smooth Muscle ◽

Molecular Mechanism ◽

Dunaliella Salina ◽

Flow Cytometric Analysis ◽

Nuclear Antigen ◽

Brdu Incorporation ◽

Dependent Manner ◽

Balloon Injury ◽

Neointimal Formation

The pathological mechanism of restenosis is primarily attributed to excessive proliferation of vascular smooth muscle cells (VSMC). The preventive effects of ethanol extract of Dunaliella salina (EDS) on balloon injury-induced neointimal formation were investigated. To explore its molecular mechanism in regulating cell proliferation, we first showed that EDS markedly reduced the human aortic smooth muscle cell proliferation via the inhibition of 5′-bromo-2′-deoxyuridine (BrdU) incorporation at 40 and 80 μg/ml. This was further supported by the G0/G1-phase arrest using a flow cytometric analysis. In an in vivo study, EDS at 40 and 80 μg/ml was previously administered to the Sprague–Dawley rats and found that the thickness of neointima, and the ratio of neointima:media were also reduced. EDS inhibited VSMC proliferation in a dose-dependent manner following stimulation of VSMC cultures with 15 % fetal bovine serum (FBS). Suppressed by EDS were 15 % FBS-stimulated intracellular Raf, phosphorylated extracellular signal-regulated kinases (p-Erk) involved in cell-cycle arrest and proliferating cell nuclear antigen. Phosphorylated focal adhesion kinase (p-FAK) was also suppressed by EDS. Also active caspase-9, caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels were increased by administration with EDS; the apoptotic pathway may play an important role in the regulatory effects of EDS on cell growth. These observations provide a mechanism of EDS in attenuating cell proliferation, thus as a potential intervention for restenosis.

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The effect of germ cell complement on the presence of oxytocin in the interstitial and seminiferous tubule fluid of the rat testis

Journal of Endocrinology ◽

10.1677/joe.0.1430471 ◽

1994 ◽

Vol 143 (3) ◽

pp. 471-478 ◽

Cited By ~ 7

Author(s):

H D Nicholson ◽

H M Greenfield ◽

J Frayne

Keyword(s):

Heat Treatment ◽

Germ Cell ◽

Germ Cells ◽

Seminiferous Tubule ◽

Leydig Cells ◽

Male Rats ◽

Rat Testis ◽

Pachytene Spermatocytes ◽

Tubule Fluid

Abstract In the rat testis oxytocin has been localized to the Leydig cells, and these cells have been shown to produce oxytocin in vitro. The present study was performed to determine whether oxytocin is present in the interstitial fluid (IF) and seminiferous tubule fluid (TF) of the rat and whether concentrations of the peptide vary within the two compartments following germ cell destruction. In order to destroy germ cells adult male rats were anaesthetized and their scrotal regions placed in a water bath at 43 °C for 20 min. Control animals were subjected to anaesthesia alone. Groups of 6 animals were killed 3, 7 and 21 days after heat treatment and their testes removed for histological examination or fluid extraction. IF and TF were separated and the oxytocin content of the fluids measured by radioimmunoassay. Immunoreactive oxytocin was detected in both the IF (100 ±11 pg/ml) and TF (27± 4 pg/ml) of control rats and this immunoreactivity co-eluted with the authentic peptide following HPLC. Three days after heat treatment IF levels of oxytocin were significantly reduced but TF levels of the peptide were significantly increased. These changes were associated with a lack of pachytene spermatocytes in the histological sections. Seven and 21 days after heat treatment the levels of oxytocin in the IF and TF were not significantly different from control levels. Similar changes in IF and TF oxytocin levels were seen in a second experiment when pachytene spermatocytes were removed using the testicular toxicant methoxyacetic acid. To investigate whether oxytocin passes from the IF into the TF, 3H-oxytocin was infused into the testicular arteries of both control and heat-treated rats and the rats killed at regular intervals over the next 24 h. In both groups of animals <5% of the 3H-oxytocin passed from the IF into the TF. These data suggest that the seminiferous tubule as well as the Leydig cells may secrete oxytocin and that this secretion may be influenced by the presence of germ cells, particularly pachytene spermatocytes. Journal of Endocrinology (1994) 143, 471–478

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