FSH Antiserum Research Articles

Effects of differential pulse frequencies of chicken gonadotrophin-releasing hormone-I (cGnRH-I) on laying hen gonadotrope responses in vitro

The aim of this work was to determine the effects of cGnRH I pulse frequencies on FSH and LH release and the changes in features and number of cultured laying hen FSH-cells and LH-cells in vitro. Primary adenohypophyseal cell cultures taken from laying hens were stimulated by four 5 min pulses using 1 or 10 nM cGnRH, administered with interpulses between pulses at 15, 30 or 60 min. Pulse frequencies and dose dependent effects were examined in six separate experiments including two controls. After the last interpulse time, the supernatants were collected and stored at −70° C until the performance of an indirect enzyme-linked immunosorbent assay (ELISA) using chicken LH and chicken FSH antisera at 1:1000 and 1:2000 dilutions, respectively. Supernatants were coated in duplicate on the inner surface of Immulon 2 plates and later blocked with the optimal solutions. They were incubated with each antiserum and subsequently with isotype-specific peroxidase-labeled anti-rabbit antibodies. Hydrogen peroxide/o-phenylenediamine was added as substrate/chromogen and the optical density (OD) was determined at 492 nm. The ABC immunocytochemical method was performed to characterize and re-count the gonadotropes employing anti-chicken FSH and anti-chicken LH as primary antibodies. The number of FSH-LH cells was obtained using stereological analysis and the data were statistically processed. The ODs obtained for each anti-hormone were compared with the control groups and with each other. Significant differences were found in number of aggregated-positive LH cells, which decreased with 1 nM cGnRH-I, 15 vs. 30 min pulses, increased with 30 vs. 60 min pulses, and also with 10 nM cGnRH-I, 30 vs. 60 min pulses. Aggregated positive FSH cells, however, did not show significant differences in percentage at any GnRH dose or pulse frequencies, but did show activity at low pulse frequencies of 15 and 30 min. The results suggest that LH cells varied in percentage in a dose dependent manner at higher pulse frequency (15 min) and were dose independent at low pulse frequency (60 min) and showed inactive features; while FSH cell numbers were unaffected showing features of activity at low pulse frequencies. High and moderate pulse frequencies of cGnRH-I (15-30 min) increased the FSH release in dose independent manner without changes in features or percentage of FSH cells. Low pulse frequency (60 min) of cGnRH-I increased LH release dose independently disminished LH cell percentage and showed changes in cells’ features. These results in avian cells showed differences in responses to GnRH pulse frequencies from those reported earlier in mammals.

Role of FSH in regulating testicular germ cell transformations in the rat: a study using DNA flow cytometry.

The role of FSH in regulating testicular germ cell transformations during initiation and maintenance of spermatogenesis in the pubertal and adult rat has been studied using DNA flow cytometry (FCM). The cell types were quantified on the basis of their DNA content using DNA specific fluorochrome DAPI (4,6-diamidino phenylindole). Pubertal (30-d old) and adult (100-d old) rats were deprived of endogenous FSH support for 10 d by daily injection (200 microliters d-1) of a characterized FSH antiserum; the control group received an equivalent volume of normal rat serum. FSH deprivation did not lead to any change in serum testosterone levels. The relative proportion of testicular germ cells in the FSH deprived pubertal rat showed a 90% reduction in 1C (round spermatids) and 260% and 90% increase in 2C (spermatogonia) and 4C (spermatocytes) cells respectively. While the overall conversion of 2C to 1C (1C:2C ratio) was reduced by 98%, the transformation of 2C to 4C (4C:2C ratio) and 4C to 1C (meiotic division 1C:4C ratio) was inhibited by 43% and 93% respectively. In the FSH-deprived adult rat the overall conversion of 2C to 1C was reduced by 26% (P < 0.05) only. The 2C and 4C population of cells increased by 47% and 97% respectively (P < 0.025) and the 4C:2C ratio by 47% (P < 0.05). While the meiotic division (1C:4C ratio) was reduced by 54% (P < 0.001), the post-meiotic differentiation of round spermatids to elongate-spermatids (HC:1C) was inhibited by 68% (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH

To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors.

Developmental expression of estrogen receptor (ER) alpha and ERbeta in the hamster ovary: regulation by follicle-stimulating hormone.

Perinatal expression of estrogen receptor (ER) protein and mRNA and the influence of FSH on this process were examined by immunofluorescence and RT-PCR using ovaries from fetal (d 13-15 of gestation) and postnatal [postnatal d 1-15 (P1-P15)] hamsters and from 8-d-old hamsters exposed in utero to an anti-FSH serum on d 12 of gestation and saline or equine chorionic gonadotropin (eCG) on P1. A few somatic cells expressing ERalpha immunoreactivity appeared first on d 14 of gestation and increased markedly by P8-P15 in the interstitial cells and granulosa cells of primordial follicles. In contrast, appreciable ERbeta immunoreactivity was localized on d 13 of gestation, and more cells expressed ERbeta immunoreactivity by P1-P8. By P7, ERbeta immunoreactivity was present in cells adjacent to the oocytes, and by P8, ERbeta was preferentially localized in the granulosa cells. Receptor immunoreactivities decreased markedly in P8 ovaries exposed in utero to the FSH antiserum but were reversed with postnatal eCG replacement. Oocytes and somatic cells expressed ERalpha and ERbeta mRNA, and levels of ER mRNA in the ovary increased by P7-P8, corresponding to the appearance of primordial follicles. Thereafter, only ERbeta mRNA levels increased progressively with postnatal ovary development. Similar to ER protein, mRNA levels decreased significantly in FSH antiserum-treated ovaries but were restored by eCG. These results indicate that both ER subtypes are expressed in undifferentiated somatic cells and the oocytes during perinatal ovary development in the hamster; however, ERbeta expression segregates with the differentiation of granulosa cells. Furthermore, ER expression and differentiation of somatic cells to granulosa cells depend on perinatal FSH action.

Growth differentiation factor-9 and stem cell factor promote primordial follicle formation in the hamster: modulation by follicle-stimulating hormone.

Growth differentiation factor-9 (GDF-9) and stem cell factor (SCF) influence follicle formation beyond the primary stage; however, factors influencing the formation of primordial follicles remain elusive. To determine whether GDF-9 and SCF promoted primordial follicle formation during ovarian morphogenesis in the hamster, and whether FSH had any modulatory influence, fetal ovaries were collected on Gestation Day 15 from pregnant hamsters treated with or without an FSH antiserum on Gestation Day 12 and cultured in vitro up to Day 9 with SCF, GDF-9, or FSH. The percentages and diameters of primordial, primary, and secondary follicles and their oocytes were determined by morphometric evaluation, and the expression of GDF-9 was detected by immunolocalization. SCF, GDF-9, and FSH promoted primordial and primary follicle formation, but GDF-9 was more efficient. The diameters of the follicles developed under GDF-9 or FSH, but not SCF, compared well with those developed in vivo. FSH- and GDF-9-induced folliculogenesis was attenuated by the SCF antibody. Similarly, in vitro formation of primordial follicles decreased markedly in ovaries exposed to the FSH antiserum in utero, which was reversed by SCF, GDF-9, or FSH; however, GDF-9 had a profound effect on follicular development. GDF-9 protein appeared exclusively in the oocytes on Postnatal Day 4; however, it appeared in vitro by 48 h, and the expression was upregulated by FSH. These results suggest that although SCF-induced primordial follicle formation constitutes primarily somatic cell development, GDF-9 influences both the oocyte and its companion somatic cells. FSH plays an important role in primordial folliculogenesis in the hamster via GDF-9 and SCF.

Development of a sensitive enzymeimmunoassay (EIA) for FSH determination in bovine plasma

A highly sensitive enzymeimmunoassay (EIA) procedure for FSH determination in bovine plasma on microtiterplates using the biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to FSH and used to bridge between streptavidin-peroxidase and the immobilized antiserum in the competitive assay. The EIA was carried out directly in 50 microl of bovine plasma and compared with an established radioimmunoassay (RIA) employing 100 microl plasma. Same FSH standards and FSH specific antiserum were used in both procedures. FSH standards prepared in hormone free plasma were used. The sensitivity of the EIA procedure was 6.25 pg/well FSH which corresponded to 125 pg/ml plasma; the 50% relative binding sensitivity was seen at 200 pg/well. In comparison to RIA, the EIA was at least four times more sensitive besides requiring 6 times less FSH specific antiserum. Plasma volumes for the EIA ranging from 12.5 to 50 microl did not influence the shape of the standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. When both EIA and RIA methods were used to measure FSH in cows, the levels were detectable only by the EIA procedure. The assay detects high and low plasma FSH levels within the physiological variation as well as changes in plasma FSH after stimulation with a GnRH analog. In conclusion, in addition to being non-radioactive and low cost in nature, the method offers several advantages over the conventional FSH RIA procedure; these are (a) higher sensitivity, (b) less labour and time saving, (c) more economical use of precious FSH antiserum and (d) long shelf-life of the biotinyl-FSH label (in contrast to the short half life of iodinated FSH in RIA).

Enhancement of FSH bioactivity in vivo using site-specific antisera.

The ability of site-specific antipeptide antisera to enhance the biological activity of ovine FSH (oFSH) in vivo was investigated using hypopituitary Snell dwarf mice. These animals were shown to respond to increasing doses of oFSH (3.3-90 micrograms/day), administered in two daily injections over a 5-day treatment period, in a highly significant dose-dependent fashion. The responses measured were increases in uterine weight, ovarian weight and the index of keratinisation in vaginal smears. The dose-dependent response to oFSH confirmed the suitability of this animal model for these investigations and suggested the suboptimal dose of oFSH (20 micrograms/day) for use in enhancement studies. Five peptides derived from the beta subunit of bovine FSH (bFSH) (A, residues 33-47; B, 40-51; C, 69-80; D, 83-94; E, 27-39) were used to generate polyclonal antipeptide antisera. Of these peptides, only A and B produced an antiserum (raised in sheep) capable of recognising 125I-bFSH in a liquid phase RIA. Antisera prepared against peptide A or peptide B were found to significantly enhance the biological activity of 20 micrograms oFSH/day over a 5-day treatment period. The response to antipeptide antisera alone did not differ significantly from that observed in PBS-injected control animals, neither did the response to FSH alone differ from that observed in animals treated with FSH plus preimmune serum. Thus the enhanced responses are dependent upon the presence of FSH plus antipeptide antiserum. Peptides A and B are located in a region thought to be involved in receptor recognition, this may have implications for the mechanism underlying this phenomenon and/or the structure/function relationships of FSH. That FSH-enhancing antisera can be generated by immunisation of animals with peptides A and B suggests that it may be possible to develop these peptides as vaccines capable of increasing reproductive performance, such as ovulation rate. The high degree of sequence homology between ovine, bovine and porcine (and to a lesser extent human and equine) FSH in the region covered by peptides A and B suggests that these peptides could also be used to promote and regulate ovarian function in all of these species.

Evidence for Follicle-Stimulating Hormone Mediation in the Hemiorchidectomy-Induced Compensatory Increase in the Function of the Remaining Testis of the Adult Male Bonnet Monkey (Macaca Radiata)1

Hemiorchidectomy (HO) in the adult male bonnet monkey results in a selective increase in circulating concentrations of FSH and testosterone, and this is accompanied by compensatory increase in sperm production by the remaining testis. We investigated the possible role of increased FSH concentration that occurs after HO in the compensatory increase in the activity of the remaining testis. Of eight adult male bonnet monkeys that underwent HO, four received i.v. injections every other day for 30 days of a well-characterized ovine FSH antiserum (a/s) that cross-reacts with monkey FSH. The remaining four males received normal monkey serum (NMS) as control treatment in a protocol similar to that employed for a/s-treated males. Blood samples were collected between 2100 and 2200 h before and 1/2, 1, 3, 5, 7, 14, 22, and 29 days after HO. Testicular weight, number of 3 beta-hydroxy steroid dehydrogenase-positive (3 beta-HSD+) cells, and DNA flow cytometric analysis of germ cell populations were obtained for testes collected before and at the termination of NMS or a/s treatment. In NMS-treated males, circulating serum FSH concentrations progressively increased to reach a maximal level by Day 7 after HO (1.95 +/- 0.3 vs. 5.6 +/- 0.7 ng/ml on Days -1 and 7, respectively). Within 30 min of a/s injection, FSH antibodies were detected in circulation, and the antibody level was maintained at a constant level between Day 7 and end of treatment (exhibiting 50-60% binding to 125I-hFSH).(ABSTRACT TRUNCATED AT 250 WORDS)

In Vivo and in Vitro Studies on the Differential Role of Luteinizing Hormone and Follicle-Stimulating Hormone in Regulating Follicular Function in the Bonnet Monkey (Macaca radiata) Using Specific Gonadotropin Antibodies1

Although a distinct need for FSH in the regulation of follicular maturation in the primate is well recognized, it is not clear how FSH controls the functionality of different cellular compartments of the follicle. It is also not evident whether there is a requirement for LH in follicular maturation in the primate. In the first part of the present study, female bonnet monkeys were administered a well-characterized ovine (o) LH antiserum to neutralize endogenous monkey LH for different periods during the follicular phase, and the effect on the overall follicular maturation process was assessed by analyzing serum estrogen (E) and progesterone (P) profiles. Neither continuous LH deprivation from Day 8 of the cycle nor deprivation of LH on any one day between Days 6 and 10 had a significant effect on serum E and P profiles and the follicular maturation process. The period for which the antiserum was effective was dependent upon the dose injected; 1 ml of the antiserum given on Day 8 blocked ovulation but not follicular maturation. To assess the effect of deprivation of LH/FSH at the cellular level, animals were deprived in vivo of LH (on Days 8 and 9 of the cycle) or of FSH (on Day 6 of the cycle) by injection of highly characterized hCG and ovine (o) FSH antisera, respectively; the in vitro responsiveness of granulosa and thecal cells isolated on Day 10 from these animals was then determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of neurotensin-immunoreactive cells in the anterior pituitary of normal and castrated rats. A double immunocytochemical investigation at the light- and electron-microscopic levels.

The localization of neurotensin (NT) has been studied in the rat pituitary by means of immunocytochemistry at the light- and electron-microscopic levels. Cells immunoreactive for NT (NT-IR) were observed in the anterior lobe of rats of both sexes. Following an elution-restaining procedure, it was found that most of these NT-IR cells correspond to cells also reacting to beta-LH or FSH antisera. However, a few NT-IR cells did not stain for beta-LH nor FSH, but for TSH. The subcellular localization of NT was studied at the electron-microscopic level by means of the immunogold procedure. The immunoreactivity was always localized over the secretory granules. By using two sizes of gold particles, it was shown that NT- and beta-LH-IR were colocalized in part of the cell granules. Castration caused a progressive decrease of the NT-IR in the beta-LH- or FSH-positive cells, while the number of NR-IR, TSH-IR cells increased. After 2 weeks, NT-IR was mainly observed in this latter cell type. The present work demonstrates that NT-IR is localized within the secretory granules of the gonadotrophs and a few thyrotrophs, indicating that NT could be released together with gonadotrophins and TSH. Moreover, since gonadectomy results in typical qualitative and quantitative changes of NT-IR gonadotrophs, the intervention of the pituitary NT in the regulation of the hypophysogonadal axis is strongly suggested.

Immunohistochemical Identification of Mouse Adenohypophysial Gonadotropes

Mouse pituitary gonadotropes were identified using immunohistochemical techniques with OsO4 single fixation. Most gonadotropes are immunostained with both LH beta and FSH antisera (FSH/LH gonadotropes), whereas a few gonadotropes are positive only for LH beta (LH gonadotropes). The former gonadotropes are large rounded or oval cells and contain two types of secretory granules: electron-dense smaller ones (about 100-200 nm in diameter) and electron-transparent larger ones (maximum diameter, about 400 nm) enclosed by a halo. The endoplasmic reticulum appears as a series of dilated sacs, and the Golgi apparatus is distinct. The mitochondria are found to have an electron-dense matrix throughout the cytoplasm, but are often concentrated in and around the region of the Golgi apparatus. These fine structural features are somewhat similar to those of the classical Barnes "FSH cell" The LH gonadotropes are polygonal cells of medium size. Small granules, as described above, are seen, but large granules are not. The Golgi apparatus is distinct, but small. The LH gonadotropes resemble neither the Barnes "LH cell" nor the Kurosumi-Oota "LH cell".

Immunohistochemical Study of Adenohypophysial Gonadotropes in the Musk Shrew, Suncus murinus L. (Insectivora)

Most gonadotropes in the pituitary of male Suncus murinus L. are immunostained with both LH beta and FSH antisera, whereas only a few gonadotropes are positive only for LH beta. The former gonadotropes consist of polygonal or polyhedral cells of medium size, and can be identified by the presence of two types of secretory granules of low density: smaller ones (about 150-250 nm in diameter) which are spherical in shape, and larger ones of irregular form with a maximum diameter of about 1,500 nm. The endoplasmic reticulum is not prominent, while the Golgi apparatus is distinct. Mitochondria are found throughout the cytoplasm but are often concentrated in and around the region of the Golgi apparatus. These fine structural features are somewhat similar to those of the classical Barnes "FSH cells" and Kurosumi-Oota "FSH cells".

Luteal-phase defect induced by deprivation of FSH at a specific period of the follicular phase prevents pregnancy in the bonnet monkey (Macaca radiata)

Female bonnet monkeys were injected i.v. with 25 microliters antiserum to FSH on Days 5, 6 or 7 of the cycle: the length of the luteal phase was shortened but there was no alteration in cycle length. Proven fertile females (N = 6) were caged throughout the period of the experiment (6 cycles) with proven fertile males and treated with 25 microliters FSH antiserum on Day 7 of each of 3 successive cycles. Out of 18 cycle exposures during the treatment phase, 17 were ovulatory, but no pregnancies occurred. In the post-treatment phase, 5 monkeys became pregnant within 3 cycle exposures. These results show that it is possible to render female monkeys infertile by creating luteal insufficiency and this can be achieved repeatedly in a reproducible manner by depriving the cyclic females of FSH support on Day 7 of consecutive cycles.

Identification of a Potential FSH Modulatory Protein in Human Testis and Seminal Plasma

A variety of factors capable of inhibiting the in vitro binding of FSH to its receptor have been identified in gonadal tissues from males and females. Interest in these factors has been stimulated because of their potential role as local modulators of gonadotropin action. Studies reported here were undertaken to determine if proteins having antigenic homologies with human FSH or an "FSH-like" protein isolated from porcine follicular fluid were present in human testicular tissue or seminal plasma. Polyclonal antibodies were generated against fractions of porcine follicular fluid containing FSH receptor binding inhibitory activity, FSH agonist activity in vitro, and a 58,000 Mr protein recognized by human FSH antiserum. Antiserum against this fraction of porcine follicular fluid and antiserum against human FSH were used to probe Western blots of proteins from human testis homogenates or seminal plasma. A 58,000 Mr protein was identified in both human testis extract and seminal plasma. This protein appears to be related antigenically to both human FSH and the 58,000 Mr "FSH-like" protein in porcine follicular fluid. It does not appear to be a metabolic degradatory product of human FSH since the protein is larger than FSH, does not dissociate into subunits under reducing conditions and is recognized by the antiserum to FSH-like protein that does not recognize human FSH. These data identify a 58,000 Mr protein in human testis and seminal plasma that may represent a local modulator of FSH action in the testis.

Effect of FSH deprivation at specific times on follicular maturation in the bonnet monkey (Macaca radiata)

Cyclic monkeys were deprived of FSH for specific periods on different days of the follicular phase by injecting them with minimal doses of an FSH antiserum characterized for specificity and bioneutralizing ability. The effect of the antiserum on follicular maturation was assessed by determining (a) serum oestrogen concentrations through the midcycle period, (b) serum progesterone concentrations as an index of ovulation and luteal function, (c) laparoscopic examination of the surface of the ovary when necessary, and (d) overall cycle length. While antiserum injection on Day 5 of the cycle caused delay in the oestrogen surge from Days 9 to 11, injection on Day 6 led to the occurrence of two oestrogen surges, on Days 9 and 14. Laparoscopic examination showed that the earlier follicle had disappeared and a new follicle had appeared by Day 14. Antiserum injection on Day 7 of the cycle arrested further growth of the maturing follicle, but a new follicle appeared 9 days later, as indicated by a surge of oestrogen on Day 16. Injection of antiserum beyond Day 7 had no effect on follicular development, ovulation and luteal function. These observations suggest that the mature follicle becomes relatively independent of FSH support about 48 h before ovulation and this event could be a marker for follicular dominance.

Immunohistochemical and ultrastructural study of anterior pituitary cells in the female Afghan pika, Ochotona rufescens rufescens.

An immunohistochemical study of the anterior pituitary gland of the female Afghan pika was carried out to distinguish the ultrastructural features of GH, PRL, ACTH, TSH and LH cells. The histochemically identified GH cells resembled ultrastructurally oval or round GH cells of the rat laden with large, dense secretory granules. PRL cells were divided into three subtypes based on differences in the diameter of their spherical secretory granules. They lacked polymorphic or irregularly shaped secretory granules. ACTH cells resembled ultrastructurally, in some respects, Siperstein's "corticotrophs" of the rat with peripheral arrangement of secretory granules. However, they were not always stellate, but elongate or angular in shape. The dense secretory granules were concentrated in the peripheral area of cytoplasm. TSH cells were non-stellate, but usually oval in shape, containing the smallest spherical secretory granules (100-200 nm in diameter). Almost all LH cells reacted also with FSH antiserum. They were irregular in shape, sometimes in contact with or surrounded the GH cells. They contained an abundance of medium-sized secretory granules (140-260 nm in diameter) which were larger than those in the LH cells of the female rat throughout the estrous cycle. Large secretory granules in the LH cells of the female pika seemed to be related to the endocrine state of persistent estrus.

Studies on follicular atresia: role of tropic hormone and steroids in regulating cathepsin-D activity of preantral follicles of the immature rat

The ovary of the immature female rat is comprised of primary and medium-sized preantral follicles. Upon stimulation with FSH or PMSG, the cathepsin-D activity, a representative lysosomal enzyme of granulosa cells, is reduced by 50% ( P < 0.01). 17β-Estradiol at the doses tried was unable to mimic this effect. Blockade of steroidogenesis with cyanoketone also had no effect on the cathepsin-D activity of isolated granulosa cells. Dihydrotestosterone (DHT), however, at a dose of 1 mg/rat was able to inhibit PMSG's tropic action. It brought about an increase in cathepsin-D activity and reduction in steroidogenic activity of isolated granulosa cells. The atretogenic activity of DHT could be relieved by supplementation with exogenous FSH. DHT was observed to significantly reduce ( P < 0.01) endogenous FSH and LH levels within 12–18 h of its injection suggesting that its atretic effect was due to its action at the pituitary rather than the gonad. In addition to the above the ability of 15 IU of PMSG to reduce cathepsin-D activity of granulosa cells was also significantly reduced ( P < 0.01) if endogenous FSH was neutralized by a specific FSH antiserum. The present study suggests that as far as small and medium-sized primary and preantral follicles are concerned, FSH lack is the essential signal for onset of atresia.

Avian LH and FSH: Comparison of several radioimmunoassays

Comparisons were made between various LH and FSH radioimmunoassays currently being used to measure avian hormones. The two LH assays were the homologous chicken system of Follett et al. (1972) and the turkey assay of Burke et al. (1979) . These assays were also used in heterologous arrangement by interchanging the iodinated LH fractions and antisera. Five FSH assays were analyzed: two homologous chicken systems ( Scanes et al., 1977 ; Sakai and Ishii, 1980), an assay based on mammalian materials (rat FSH and anti-ovine FSH; Follett, 1976), a heterologous system using labelled chicken FSH with the anti-ovine FSH antiserum, and one using labelled turkey FSH (Burke et al., 1979) with an anti-chicken FSH antiserum. The potencies of purified chicken and turkey gonadotrophin preparations and of a range of plasma samples from Japanese quail were measured in each assay. The two LH systems showed some degree of species specificity, such that chicken LH was more active than turkey LH in the chicken assay, whereas the reverse was true in the turkey LH assay. The potency estimates of the purified hormones in the various FSH assays were very consistent. The qualitative changes in plasma hormone levels were similar in all assays, although there were some differences in the magnitude of the responses.

A whole range of fine structural criteria for immunohistochemically identified LH cells in rats.

Pituitaries from normal, young and adult male rats were fixed either in sublimate-formalin or in glutaraldehyde-osmium. In adjacent Paraplast sections, almost all the gonadotrophs were immunostained with both LH and FSH antisera. The rat LH beta and FSH antisera used were shown to be highly specific by the absorption test and by double antibody radioimmunoassay. Thin and thick adjacent Epon sections were prepared for EM and immunohistochemical examination. Cells stained with the rat LH beta antiserum were identified by LM, and the observed in detail by EM. On the basis of these observations we suggest that the LH cells are arranged in a sequence of basophils, i.e., Types II/III, III, III/IV and IV: Type II/III basophils are elongate with a cytoplasmic process and less vesiculated. They have morphological features of Type II (classical thyrotrophs) and also of Type III basophils. Type III basophils are oval in shape and moderately vesiculated. Both Types II/III and III basophils can be divided into two classes of cell characterized mainly by the existence of only small secretory granules (150-220 nm in diameter) (Type A) or by the coexistence of small and large (350-500 nm) (Type B). Type III/IV basophils are cells intermediate between types III and IV basophils, and moderately vesiculated with an abundance of secretory granules (150-300 nm in diameter). Type IV basophils are large, spherical or oval cells whose RER cisternae are conspicuously dilated; they contain less numerous secretory granules (150-300 nm in diameter). It is concluded that LH cells are not a single cell type, but include a wide range of subtypes.

Characterization of a radioimmunoassay for ovine FSH utilizing an anti-bovine FSH serum.

The sensitivity of the assay, utilizing a bovine FSH antiserum and 125I-labelled bovine FSH tracer, is 5 ng NIH-FSH-S6/ml, and cross-reactivities of other ovine pituitary glycoprotein and protein hormones (LH, TSH, growth hormone and prolactin) are less than 1.0%. Both pituitary and serum FSH of the sheep cross-react in a manner parallel with that of standard NIH-FSH-S6. Serum FSH concentrations in 4 ewes at various times after the onset of oestrus peaked more or less coincidently with the preovulatory increase of serum LH. The serum levels of FSH and LH did not correlate proportionally but fluctuated considerably in individuals animals. The radioimmunoassay system appears to be sensitive and specific for measuring serum concentrations of FSH in rams and ewes in different physiological states.