Mammalian Follicle-stimulating Hormone Research Articles

In Silico Perspectives on Gonadotropin Crosstalk

The pivotal role of the gonadotropins (GtHs) luteinizing hormone (LH) and follicle stimulating hormone (FSH) in vertebrate reproduction is well documented. LH and FSH bind to the leucine-rich repeats of the extracellular domains of their cognate receptors (R) in the gonads to actuate steroidogenesis and gametogenesis. Though these GtHs specifically bind only to their cognate receptors in mammals, this interaction becomes inconsistent in the case of fish. Whilst in some fish species the gonadotropins show receptor specificity much like their mammalian homologs, FSH and LH have demonstrated mutual and/or singular cross-activation of the FSH and/or LH receptors in several fish species. These complications regarding receptor specificity are further magnified by cross activation by orthologous GtHs from different species. So far no consistent pattern has been established to chart or predict this cross-talk in specific species or higher taxonomic orders. In the current study, we strived to understand the promiscuous nature of FSH and LH through in silico perspectives. While our studies in Russian sturgeon (Acipenser gueldenstaedtii) have shown FSH and LH to exhibit mutual promiscuity but of varying magnitude, in common carp (Cyprinus carpio) only LH showed singular cross-activation of FSHR. In Nile tilapia (Oreochromis niloticus) these gonadotropins exhibited strict specificity towards their cognate receptors similar to humans. Taking these species as representatives of varying degrees of cognate binding shown by the gonadotropins, we have generated docked models of these hormones and the extracellular domain of these receptors using in silico tools. We have further tried to compare these models, their docked poses, and key residues involved in the binding of these hormone-receptor complexes. We have further tried to compare parameters such as phosphorylation sites, N-linked glycosylation motifs, surface charge, etc. of these models to better understand the docking of these complexes. Our results suggest that in cases of cross activation by FSH and/or LH within or across the species, though binding sites are present at the leucine rich repeats on concaved face of the receptor extracellular domain, the docked position and residues involved in binding may vary significantly. Furthermore, though the seatbelt loop and GTHβ loop-2 (gonadotropin hormone subunit- beta) of the gonadotropin hormones act as the key players in binding, the docking of the hormone may vary in conformation. Due the large size and flexibility of these hormones and the receptor binding interface, it seems apparent that there are multiple binding sites that activate the resulting signaling pathway. Our study aspires to provide better insights towards the promiscuous nature of these hormones and attempt to overcome the handicap of lack of individual physical models using in silico tools.

Advances in the Regulation of Mammalian Follicle-Stimulating Hormone Secretion.

Simple SummaryThe reproduction of mammals is regulated by the hypothalamic-pituitary-gonadal axis. Follicle stimulating hormone, as one of the gonadotropins secreted by the pituitary gland, plays an immeasurable role. This article mainly reviews the molecular basis and classical signaling pathways that regulate the synthesis and secretion of follicle stimulating hormone, and summarizes its internal molecular mechanism, which provides a certain theoretical basis for the research of mammalian reproduction regulation and the application of follicle stimulating hormone in production practice.Mammalian reproduction is mainly driven and regulated by the hypothalamic-pituitary-gonadal (HPG) axis. Follicle-stimulating hormone (FSH), which is synthesized and secreted by the anterior pituitary gland, is a key regulator that ultimately affects animal fertility. As a dimeric glycoprotein hormone, the biological specificity of FSH is mainly determined by the β subunit. As research techniques are being continuously innovated, studies are exploring the underlying molecular mechanism regulating the secretion of mammalian FSH. This article will review the current knowledge on the molecular mechanisms and signaling pathways systematically regulating FSH synthesis and will present the latest hypothesis about the nuclear cross-talk among the various endocrine-induced pathways for transcriptional regulation of the FSH β subunit. This article will provide novel ideas and potential targets for the improved use of FSH in livestock breeding and therapeutic development.

Follicle-stimulating hormone receptor (FSHR) alternative skipping of exon 2 or 3 affects ovarian response to FSH

Genes critical for fertility are highly conserved in mammals. Interspecies DNA sequence variation, resulting in amino acid substitutions and post-transcriptional modifications, including alternative splicing, are a result of evolution and speciation. The mammalian follicle-stimulating hormone receptor (FSHR) gene encodes distinct species-specific forms by alternative splicing. Skipping of exon 2 of the human FSHR was reported in women of North American origin and correlated with low response to ovarian stimulation with exogenous follicle-stimulating hormone (FSH). To determine whether this variant correlated with low response in women of different genetic backgrounds, we performed a blinded retrospective observational study in a Turkish cohort. Ovarian response was determined as low, intermediate or high according to retrieved oocyte numbers after classifying patients in four age groups (<35, 35-37, 38-40, >40). Cumulus cells collected from 96 women undergoing IVF/ICSI following controlled ovarian hyperstimulation revealed four alternatively spliced FSHR products in seven patients (8%): exon 2 deletion in four patients; exon 3 and exons 2 + 3 deletion in one patient each, and a retention of an intron 1 fragment in one patient. In all others (92%) splicing was intact. Alternative skipping of exons 2, 3 or 2 + 3 were exclusive to low responders and was independent of the use of agonist or antagonist. Interestingly, skipping of exon 3 occurs naturally in the ovaries of domestic cats--a good comparative model for human fertility. We tested the signaling potential of human and cat variants after transfection in HEK293 cells and FSH stimulation. None of the splicing variants initiated cAMP signaling despite high FSH doses, unlike full-length proteins. These data substantiate the occurrence of FSHR exon skipping in a subgroup of low responders and suggest that species-specific regulation of FSHR splicing plays diverse roles in mammalian ovarian function.

Promotion of spermatogonial proliferation by neuregulin 1 in newt (Cynops pyrrhogaster) testis

We have previously shown that mammalian follicle-stimulating hormone (FSH) promotes the proliferation of spermatogonia and their differentiation into primary spermatocytes in organ culture of newt testis. In the current study, we performed microarray analysis to isolate local factors secreted from somatic cells upon FSH treatment and acting on the germ cells. We identified neuregulin 1 (NRG1) as a novel FSH-upregulated clone homologous to mouse NRG1 known to control cell proliferation, differentiation and survival in various tissues. We further isolated cDNAs encoding two different clones. Amino acid sequences of the two clones were 75% and 94% identical to Xenopus leavis immunoglobulin (Ig)-type and cysteine-rich domain (CRD)-type NRG1, respectively, which had distinct sequences in their N-terminal region but identical in their epidermal growth factor (EGF)-like domain. Semi-quantitative and quantitative PCR analyses indicated that both clones were highly expressed at spermatogonial stage than at spermatocyte stage. In vitro FSH treatment increased newt Ig-NRG1 (nIg-NRG1) mRNA expression markedly in somatic cells, whereas newt CRD-NRG1 (nCRD-NRG1) mRNA was only slightly increased by FSH. To elucidate the function of newt NRG1 (nNRG1) in spermatogenesis, recombinant EGF domain of nNRG1 (nNRG1-EGF) was added to organ and reaggregated cultures with or without somatic cells: it promoted spermatogonial proliferation in all cases. Treatment of the cultures with the antibody against nNRG1-EGF caused remarkable suppression of spermatogonial proliferation activated by FSH. These results indicated that nNRG1 plays a pivotal role in promoting spermatogonial proliferation by both direct effect on spermatogonia and indirect effect via somatic cells in newt testes.

Basic properties and annual changes of follicle-stimulating hormone receptors in the testis of horseshoe bats, Rhinolophus ferrumequinum.

The unique reproductive patterns, delayed fertilization in females, and asynchrony between spermatogenesis and mating behavior in males are well documented in bats living in temperate latitudes. The present study was undertaken to examine follicle-stimulating hormone (FSH) receptors in the testis of bats, Rhinolophus ferrumequinum, during the annual reproductive cycle. Male bats were captured at natural roosting sites and testicular preparations were subjected to a radioligand binding assay for FSH receptors. The weight of paired testes increased considerably in the spermatogenic period and decreased from the mating to hibernation periods. Meiotic division in the testis was observed in the spermatogenic period but not the mating period. Serum testosterone concentrations increased in the spermatogenic period and rapidly decreased in the mating period. The binding of FSH was specific for mammalian FSHs and detected primarily in the testis. Scatchard plot analyses of the binding of FSH to bat testicular preparations showed straight lines, suggesting the presence of a single class of binding sites. The affinities (equilibrium association constant) of FSH receptors were consistent throughout the annual reproductive cycle. The specific binding per unit weight of testis and total binding in the paired testes were highest in the mating period and in the spermatogenic period, respectively, among reproductive periods. The accumulation of cyclic adenosine 3', 5'-monophosphate to FSH stimulation was higher in the spermatogenic period than in the hibernation period. These findings suggest that testicular function of bats is associated with seasonal changes in the number of binding sites, while the number per target cell and the activation of adenylate cyclase led by FSH-receptor complex considerably decreases in the hibernation period.

Molecular cloning, sequence and expression of follicle-stimulating hormone receptor in the lizard Podarcis sicula

The present paper reports the full nucleotide sequence of a cloned cDNA prepared from RNA of lizard ovaries. The open reading frame consists of 2019 nucleotides, which encodes a protein of 673 amino acids belonging to the G protein-coupled receptor superfamily with a large extracellular N-terminal domain involved in hormone recognition. The transmembrane domain ends with a short intracytoplasmic COOH-terminal domain involved in effector activation. Phylogenetic analysis showed that the lizard receptor belongs to the family of follicle-stimulating hormone (FSH) receptors. The hydrophobicity profile is similar to that observed for mammalian and avian FSH receptors. Northern blot analysis of total RNA revealed that the FSH receptor is expressed at high levels in the ovary. In situ hybridization experiments demonstrate that FSH receptor mRNA is specifically localized within the small cells of the follicular epithelium surrounding the oocyte.

Mammalian follicle-stimulating hormone and insulin-like growth factor I (IGF-I) up-regulate IGF-I gene expression in organ culture of newt testis.

We previously showed that porcine follicle-stimulating hormone (pFSH) and human recombinant insulin-like growth factor (rhIGF-I) promote the differentiation of secondary spermatogonia into primary spermatocytes in organ cultures of newt testes, respectively. To elucidate the molecular action of FSH and IGF-I, we cloned cDNAs for newt IGF-I and IGF-I receptor (IGF-IR), and examined their mRNA expression in organ culture during newt spermatogenesis. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that IGF-I mRNA was highly expressed in somatic cells (mostly Sertoli cells) at the secondary spermatogonial stage but barely in germ cells, and that IGF-IR mRNA was expressed in both germ and somatic cells at all stages examined. The addition of pFSH to newt testis markedly increased IGF-I mRNA expression. Also, rhIGF-I increased IGF-I mRNA expression, whereas IGF-IR mRNA expression declined slightly. These results suggest that the ability of FSH to promote the differentiation of secondary spermatogonia is at least partly mediated by somatic cell-derived IGF-I, and that IGF-I mRNA expression in somatic cells is auto-upregulated.

Molecular Cloning, Functional Characterization, and Gene Expression of a Follicle-Stimulating Hormone Receptor in the Testis of Newt Cynops pyrrhogaster

We previously demonstrated in vitro that mammalian follicle-stimulating hormone (FSH) stimulates the proliferation of newt secondary spermatogonia and their differentiation into primary spermatocytes. In the current study, we isolated a cDNA from newt testis that encodes a FSH receptor (FSH-R). The total sequence homology in the deduced protein of the newt was approximately 70% with mammalian FSH-Rs. Mammalian cells, transiently transfected with the cloned newt FSH-R cDNA, displayed specific binding to [125I] human FSH and cAMP accumulation, indicating that the cloned cDNA encodes a functional newt FSH-R protein. Northern blot analysis revealed a single transcript of approximately 3.0 kb length that was synthesized in testicular somatic cells (mainly Sertoli cells) from spermatogonial to spermatid stages with the highest level expressed during the primary spermatocytes stage. These results demonstrate that FSH stimulates newt spermatogenesis through the FSH-R. This study, as far as we know, reports for the first time the cloning of an amphibian FSH-R cDNA.

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix.

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.

Promotion of differentiation of newt primary spermatocytes into spermatids by mammalian FSH via Sertoli cells.

Our previous studies showed that mammalian follicle-stimulating hormone (FSH) promoted the differentiation of primary spermatocytes into elongated spermatids in organ culture of testes fragments from Cynops pyrrhogaster. To elucidate the mechanism of FSH action, in this study the testes in the primary spermatocyte stage were dissociated and fractionated into germ and somatic cells, the latter comprising more than 80% Sertoli cells. Radioreceptor assays showed that FSH bound to somatic cells, very probably Sertoli cells, but not to germ cells. FSH elevated intracellular cyclic AMP levels in somatic but not germ cells. In cultures of cell aggregates somatic cells stimulated the differentiation of primary spermatocytes into round spermatids even in the absence of FSH, but to a greater extent in the presence of FSH. However, in the absence of somatic cells the extent of differentiation was similar, irrespective of the presence of FSH. Luteinizing hormone (LH) had no stimulatory effects. Most, if not all, of the somatic cells adherent to germ cells were Sertoli cells based on the criterion that they contained lipid droplets. This indicates that FSH stimulates the differentiation of primary spermatocytes via Sertoli cells. To examine if direct contact between Sertoli cells and germ cells is required for the promotion of differentiation, Sertoli and germ cells were cultured in two different compartments which were separated by a permeable membrane. Under these conditions Sertoli cells did not promote the differentiation of primary spermatocytes either in the absence or presence of FSH, indicating that direct contact between germ and Sertoli cells is required for Sertoli cells to promote the differentiation of primary spermatocytes.

Proliferation of newt spermatogonia by mammalian FSH via Sertoli cells in vitro

Our previous studies showed that mammalian follicle-stimulating hormone (FSH) stimulated the proliferation and differentiation of secondary spermatogonia into primary spermatocytes in organ culture of testes fragments from Cynops pyrrhogaster. To elucidate how FSH stimulates spermatogonial proliferation, we studied the interaction of spermatogonia with somatic cells in the presence or absence of FSH in cultures of aggregated cells derived from fractionated cell populations. When spermatogonia or those with somatic cells were cultured, they formed aggregates with themselves or with somatic cells, respectively. Most of the somatic cells in the aggregates were Sertoli cells, judged by the lipid droplets in their cytoplasm. [3H]thymidine incorporation into aggregates and autoradiography demonstrated spermatogonial proliferation that was enhanced in the presence of FSH and somatic cells (most probably Sertoli cells), but not in the absence of FSH or in the presence of luteinizing hormone (LH). To examine whether direct contact between spermatogonia and Sertoli cells is indispensable for the stimulation of spermatogonial proliferation by Sertoli cells, the two cell types were separated by a 0.4 micron pore filter in two compartments of a bicameral chamber. In this case, Sertoli cells did not stimulate spermatogonial proliferation in the presence of FSH, indicating that direct contact between spermatogonia and Sertoli cells is indispensable for the stimulation of spermatogonial proliferation by Sertoli cells. Finally, Sertoli cells isolated from the testes from the primary spermatocyte stage did not stimulate spermatogonial proliferation in the presence of FSH. This result indicated that the function of Sertoli cells with respect to their stimulation of proliferation was stage-specific.

Differentiation of Primary Spermatocytes to Elongated Spermatids by Mammalian FSH in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster: (primary spermatocytes/spermatid nuclear elongation/meiosis/newt/mammalian FSH).

Our previous studies (10, 11) showed that mammalian follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the initiation and promotion of spermatogenesis from secondary spermatogonia to primary spermatocytes in organ culture of testes fragments from the newt, Cynops pyrrhogaster. The present study demonstrated that FSH promoted in the same model system the differentiation of primary spermatocytes even further: to the stage of elongated spermatids. When testes fragments, consisting of somatic cells and germ cells (mostly primary spermatocytes), were cultured in a control medium for three weeks, only round spermatids and spermatogonia were observed; both the diameter of the cysts and the viability of the germ cells decreased to about 10-15% of the original level. On the other hand, when the medium was supplemented with FSH, elongated spermatids appeared by the second week; both the diameter of the cysts and the viability of the germ cells were maintained at a higher level than in the control medium. The effect of FSH was dose-dependent. However, neither transferrin, androgens (testosterone and 5α-dihydrotestosterone) nor luteinizing hormone (LH) was effective. The addition of cyanoketone, a specific inhibitor of 3β-hydroxy-Δ5 -steroid dehydrogenase (3β-HSD) (32), to the FSH-containing medium did not prevent the differentiation promoted by FSH, indicating that it is unlikely that Δ4 -steroid metabolites produced in fragments by FSH acted directly on germ cells. Insulin was found to improve the viability of germ cells during a 2 week of culture period. In the presence of FSH, the cells in various differentiative stages had morphological characteristics very similar to those in vivo, whereas in the absence of FSH primary spermatocytes showed abnormal features in their nuclei and cytoplasm, indicating that they were deteriorating. These results and our previous results (1-3) suggest that FSH promotes primary spermatocytes to differentiate into elongated spermatids probably by stimulating Sertoli cells to secrete factors which then act on the germ cells.

Initiation and stimulation of spermatogenesis in vitro by mammalian follicle-stimulating hormone in the Japanese newt, Cynops pyrrhogaster.

In order to elucidate the molecular mechanisms by which spermatogenesis is regulated, especially the roles of hormones and somatic cells in the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments rich in secondary spermatogonia were cultured in control medium for three weeks, most of the testicular cysts still remained as secondary spermatogonia. On the other hand, in the medium supplemented with follicle-stimulating hormone (FSH) alone, DNA syntheses in secondary spermatogonia and Sertoli cells were stimulated and secondary spermatogonia differentiated into primary spermatocytes (zygotene-pachytene) in more than half of the cysts by the second week. When newt testes fragments rich in primary spermatocytes were cultured in a control medium for three weeks only round spermatids were observed at the most advanced stage. On the other hand, in the medium supplemented with FSH alone, elongated spermatids appeared by the second week. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5 alpha-dihydrotestosterone) to the control medium stimulated differentiation for either step. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH for either stage of the testes (secondary spermatogonia and primary spermatocytes). Preliminary results indicate that FSH does not bind to germ cells but to somatic cells (most probably Sertoli cells). These and our unpublished data suggest that FSH triggers proliferation and differentiation of spermatogonia into elongated spermatids by acting on Sertoli cells which in turn act on germ cells.

Mammalian follicle-stimulating hormone stimulates DNA synthesis in secondary spermatogonia and Sertoli cells in organ culture of testes fragments from the newt,Cynops pyrrhogaster

We previously showed in organ culture of testes fragments from Cynops pyrrhogaster that mammalian follicle-stimulating hormone (FSH) stimulates secondary spermatogonia to differentiate into primary spermatocytes. In this report, we demonstrate in organ culture that FSH stimulates DNA synthesis in secondary spermatogonia and Sertoli cells: the numbers of secondary spermatogonia and Sertoli cells incorporating 5-bromo-2'-deoxyuridine (BrdU) throughout the culture period in the presence of FSH were 3-5 times those incorporating BrdU in the absence of FSH. Moreover, addition of FSH to testes fragments which had become quiescent after a week of culture in the absence of FSH, induced after a day a remarkable increase in the number of spermatogonia and Sertoli cells incorporating BrdU. The above results indicate that FSH stimulates and induces DNA synthesis in spermatogonia and Sertoli cells. Most of the spermatogonia within a cyst were labelled simultaneously and at the same density, indicating that they underwent synchronous DNA synthesis, whereas all the Sertoli cells within a cyst were not labelled simultaneously, indicating that they synthesised DNA asynchronously. When testes fragments pulse-labelled with BrdU were cultured in FSH for 14 days, the secondary spermatogonia differentiated into primary spermatocytes, whereas in the absence of FSH they failed to differentiate and most died by the 7th day. The above results together show that FSH is required for the proliferation of both secondary spermatogonia and Sertoli cells as well as the differentiation of secondary spermatogonia into primary spermatocytes.

Differentiation of Secondary Spermatogonia to Primary Spermatocytes by Mammalian Follicle-Stimulating Hormone in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster.

In order to elucidate essential factors responsible for the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments, consisting of somatic cells and germ cells almost exclusively secondary spermatogonia, were cultured in control medium for three weeks, most of the testicular cysts still contained only secondary spermatogonia. On the other hand, in the medium supplemented with various kinds of hormones and vitamins primary spermatocytes (zygotene-pachytene) appeared in about 60% of the cysts by the second week. Selective removal of specific hormones and vitamins revealed that follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the differentiation of secondary spermatogonia to primary spermatocytes. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5α-dihydrotestosterone) to the control medium stimulated differentiation. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH. Since our ultrastructural studies revealed a major loss of contact between spermatogonia and Sertoli cells following exposure to FSH, we suggest that FSH triggers differentiation of spermatogonia by acting on Sertoli cells which in turn act on spermatogonia.

Annual changes in the binding of follicle-stimulating hormone to gonads and plasma gonadotropin concentrations in Indian weaver birds inhabiting the subtropical zone

In birds, annual changes in gonadal weight are much more pronounced in the male than in the female. To analyze the mechanism inducing such a sex difference in the gonadal responsiveness to natural environmental conditions, we measured annual changes in the binding of follicle-stimulating hormone (FSH) to the gonads and the plasma gonadotropin concentration in adult Indian weaver birds inhabiting the subtropical zone. The binding of FSH was highly specific for mammalian FSHs and located primarily in the gonad. The testicular weight and FSH binding showed marked changes during the annual breeding cycle. The testicular weight was maximal in the breeding phase (June, 1987) and minimal in the nonbreeding phase (December, 1987). FSH binding per unit testicular weight was-greatest in the nonbreeding phase, while the total FSH binding per two testes was maximal in the breeding phase and minimal in the regressive phase (October, 1987). In contrast, the changes in the ovarian weight and FSH binding to the ovary were less pronounced than those in the testis. Although FSH binding per unit ovarian weight showed a peak in the nonbreeding phase; there was no significant change in the total FSH binding per ovary during the year, indicating the presence of a clear sex difference in the total FSH binding. Scatchard plot analyses of the binding suggested that the dissociation constant (Kd) ranged from 0.35 to 1.53 nM regardless of sex and season and that the changes in FSH binding were due to changes in the number of binding sites. Plasma FSH and luteinizing hormone (LH) levels markedly changed during the year in both sexes. Both FSH and LH levels were maximal in the breeding phase and minimal in the nonbreeding phase regardless of sex, although. the rate of change in either hormone tended to be greater in the male than in the female.

Properties of follicle-stimulating hormone receptors and changes during annual breeding cycle in the testis of short-tailed bandicoot rat, Nesokia indica

Some properties and seasonal changes of follicle-stimulating hormone (FSH) receptors in the testis of short-tailed bandicoot rat, Nesokia indica, were studied. The binding of FSH was highly specific for mammalian FSHs and located primarily in the testis. The Scatchard plot analyses of the binding of FSH to the testicular preparations of N. indica showed straight lines similar to those of albino Wistar rats, suggesting the presence of a single class of FSH-binding sites. The mean dissociation constants ( K d s) for FSH receptors of N. indica were 1.416 (0.964 – 2.667, 95% confidence interval) n M in December, 1986; 1.348 (0.885 – 2.849) n M in April, 1987; and 3.039 (1.678 – 16.127) n M in August to October, 1987. No significant differences were found among the three groups, but they were all significantly greater than those in Wistar rats. The FSH binding per unit weight of tissue during nonbreeding phase (June, 1987) was lower than those during other phases (December, 1986; April and August–October, 1987). Calculated numbers of FSH-binding sites showed no statistically significant differences among the latter three phases. In June preparations the number of binding sites was not determined due to their extremely low binding capacity.

Subunits of an avian (ostrich) follicle-stimulating hormone and their hybridization with subunits of mammalian gonadotropins

Follicle-stimulating hormone (FSH) from the ostrich, Struthio camelus, was dissociated by methods previously used to prepare subunits from mammalian gonadotropins. Two chemically dissimilar subunits were obtained from the ostrich FSH and these resembled the α- and β-subunits of mammalian FSH in amino acid composition. The subunits were relatively inactive in radioimmunoassay, radioreceptorassay, and bioassay when tested alone, but significant activity was regenerated upon their recombination. Incubations of mixtures of one of the ostrich subunits with the opposing subunit of ovine FSH also resulted in a significant regeneration of binding and biological activity in FSH assays. These hybrid recombinants demonstrated that the species specificity of the FSH molecule is conferred entirely by the source of the β-subunit; in fact, hybrids formed between ovine LH-α and either ovine or ostrich FSH-β were more potent in FSH assays than were those containing even two homologous FSH subunits. In contrast, there was little regeneration of LH bioactivity when FSH subunits (from ostrich or sheep) were combined with the subunits of ovine LH, although some LH binding activity was obtained when the mixture contained either one of the LH subunits; i.e., even LH-α + FSH-β showed significant enhancement of binding activity for LH receptors. Thus, the subunits of avian FSH show biochemical and functional homologies to those of mammalian FSH, but there is only limited interchangeability with ovine LH subunits.

Evolutionary and Functional Aspects of Pituitary Gonadotropins in the Green Turtle,CheloniaMydas

Information on the pituitary gonadotropins in Chelonia mydas represents some of the most complete data available for any reptile and thus provides an important basis for evaluating evolutionary processes in tetrapod endocrine physiology. The two gonadotropins isolated from Chelonia pituitary glands show clear chemical and immunological homologies to mammalian follicle-stimulating hormone (FSH) and luteinizing hormone (LH). However, receptor studies and biological tests indicate that the functions of these hormones may be different in turtles and mammals. In particular, Chelonia LH shows an unusual ability to interact with FSH receptor sites and to stimulate physiological functions normally attributed to FSH. Results with Chelonia LH demonstrate that errors may arise from using mammalian hormones to investigate reproduction in turtles. Measurements of endogenous gonadotropin levels in the plasma of breeding and nesting Chelonia provide a different perspective of the potential roles of the FSH and LH from that obtained in physiological tests. In particular, FSH and LH secretion are markedly dissociated during the nesting cycle; FSH has only a transient peak during oviposition, whereas LH, along with progesterone, displays a pronounced “ovulatory” surge in the day following nesting. Preliminary studies with synthetic gonadotropic releasing factors in Chelonia suggest that these may be useful in inducing reproductive changes.

Studies on the purification and properties of gonadotrophin from ventral lobes of the pituitary gland of the dogfish (Scyliorhinus canicula L.).

Abstract Gonadotrophin (GTH) was purified, for the first time from a cartilaginous fish by preparing a crude glycoprotein fraction of the ventral lobes of the pituitary glands of the dogfish and submitting this to affinity chromatography on concanavalin A (Con A)-Sepharose followed by ion-exchange chromatography on CM-cellulose and DEAE-cellulose. Thyroidstimulating hormone (TSH), also a glycoprotein, was purified along with the GTH. No positive attempt was made to separate the two activities, although some separation did seem to occur on Con A-Sepharose. Unlike the two teleost gonadotrophins so far tested, the dogfish material was very potent in stimulating steroidogenesis by avian testicular cells and in promoting in vivo 32 P uptake by chick testes. Although chromatography on CM-cellulose produced two peaks of GTH activity, their biological activities were similar and both fractions behaved as does mammalian follicle-stimulating hormone (FSH) on DEAE-cellulose. Only ventral lobe gonadotrophin was purified in this study, this lobe being the main source of gonadotrophin in the dogfish pituitary. The results indicate that only a single gonadotrophin was purified, though they do not prove conclusively that only one gonadotrophin is present in the dogfish pituitary.