Immunocytochemical evidence for production of luteinizing hormone and follicle-stimulating hormone in separate cells in the bovine.

In all mammalian females, follicular growth and maturation are essentially dependent on the pituitary gonadotropins, FSH and LH. These glycoprotein hormones have many similarities, but their action, based on high affinity binding to specific membrane receptors, are quite different. The purpose of this study was to perform a sensitive localization of FSH and LH in secretory granules of gonadotrophs using highly specific antisera. This morphological study included light microscopy (PAP) and electron microscopy (immunogold single and double labeling) procedures. Histologically, approximatively 11.5% of cells were positive for LH, whereas only 5.4% of cells were positive for FSH. With the electron microscope, single labeling allowed identification of morphologically distinct LH-containing cells and FSH-containing cells. Double immunostaining confirmed that no cells contained both hormones. The finding that FSH and LH are produced in separate pituitary cells is in agreement with recent studies that have suggested a specific role and regulatory process for gonadotropins in the bovine species.     

Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins

Immature rat ovaries increase their secretion of estradiol (E₂) when stimulated by gonadotropins but only after a lag period of several hours. Once established, estrogen secretion can be maintained, or increased, by the continued presence of gonadotropin. A combination of ovine FSH+LH given at 2 hr intervals stimulated the estrogen synthesizing system ($\text{ESS}$) of the ovary and serum E₂ showed a pronounced rise between 16 and 20 hrs after the initial injection. When given every 2 hrs for 5 doses (0–8 hrs) serum E₂ was undetectable. However, it was increased if 20 IU PMS was injected at the time of the last dose of FSH+ LH. Endogenous FSH&LH, increased by hourly injections of LH-releasing hormone for a period of 8 hrs, stimulated the $\text{ESS}$; serum E₂ increased at the expected time when this treatment was followed by an injection of PMS.Anti-PMS antiserum given 12 hrs after PMS, prevented the expected rise in serum E₂ at 24 hrs. However, FSH, LH or a combination of the two given every 2 hrs beginning at the time of the anti-PMS produced an increase in E₂ secretion; the combination was more effective than either hormone alone.These results are consistent with the interpretation that a combined FSH-LH action is responsible for induction of the $\text{ESS}$ in the immature rat ovary. The combination of hormones is also very effective in maintaining estrogen secretion but some function appears possible with FSH or LH alone.     

Channel Catfish Follicle-Stimulating Hormone and Luteinizing Hormone: Complementary DNA Cloning and Expression During Ovulation

Gonadotropins are important regulators of reproduction. To develop molecular resources for production of recombinant gonadotropins, we have cloned and sequenced complementary DNA encoding the channel catfish (Ictalurus punctatus) follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which encode 132 and 140 amino acid proteins, respectively. The deduced amino acid sequences of the catfish FSH and LH are highly conserved with those cloned from other teleosts. Both the FSH and the LH were highly induced during ovulation after injection of carp pituitary extract. Taken together with our previous findings of enhanced expression of growth hormone and other pituitary hormones, this research suggests that a hormonal cocktail may be needed for more efficient manipulation of catfish reproduction. The availability of the catfish FSH and LH cDNAs provides the opportunity for production of immunologically or biologically active recombinant gonadotropins for the study of catfish reproductive physiology and the manipulation of artificial spawning for aquaculture. Received March 12, 2001; accepted June 27, 2001     

Induction of ovulation and pregnancy in captive cottontail rabbits by exogenous gonadotrophin treatment and artificial insemination.

Attempts were made to induce ovulation in confined cottontail rabbits, $\text{Sylvilagus floridanus}$, by treatment with FSH and LH. FSH given twice daily for 3 days with LH on the 4th day induced ovulation. LH at 50 mg/kg bodyweight was significantly (P<0.05) more effective than at 25 mg/kg bodyweight in inducing ovulation. Daily doses of FSH in the amount of 0.05 mg to 0.20 mg induced follicular development which resulted in normal ovulation rates for the cottontail rabbit. Insemination of artifically ovulated rabbits with epididymal spermatozoa resulted in pregnancy in three of nine rabbits and in ovum fertilization in one of three rabbits.     

Periodic fluctuations in FSH concentrations during the ovine estrous cycle

Heterologous radioimmunoassays for ovine LH and ovine FSH were validated and used to examine the concentrations of gonadotropins during the estrous cycle. Concentrations of LH were maximal on the day of estrus as previously reported. Concentrations of FSH were minimal 1 or 2 days before estrus, increased markedly during estrus, and fluctuated widely during diestrus. Most ewes ($\text{11}\text{13}$) had periodic waves of FSH occurring at short intervals (3.5–6 days, $\text{3}\text{13}\text{ewes}$), long intervals (10–18 days, $\text{3}\text{13}\text{ewes}$), or at both long and short intervals ($\text{5}\text{13}\text{ewes}$).     

Phylogenetic analysis of the vertebrate glycoprotein hormone family including new sequences of sturgeon (Acipenser baeri) beta subunits of the two gonadotropins and the thyroid-stimulating hormone

The beta subunits of the two gonadotropins (GTH1 and GTH2) and of the thyroid-stimulating hormone (TSH) of a chondrostean fish, Acipenser baeri, were cloned. These new sequences and selected representative members of beta subunits of vertebrate glycoprotein hormones, including tetrapod follicle-stimulating hormones (FSH) and luteinizing hormones (LH), allowed us to infer the phylogenetic relationships within this family. Both distance matrix and maximum parsimony methods were used on both nucleotide and amino acid sequences, with bootstrapping evaluation over 1000 replicates. The four trees obtained had highly similar topologies. In each case, three monophylogenetic lineages, TSH, GTH1-FSH, and GTH2-LH were clearly identified. The three monophylogenetic lineages were supported by 21-23 specific characters at the amino acid level, out of a total of 121 characters. The resolved topologies within each monophyletic hormone cluster were congruent with the known phylogenetic relationships between the related species. The inferred parental relationships within gonadotropins are in agreement with data concerning their biological functions. The present study demonstrates that GTH1 and GTH2 are the actinopterygian homologues of tetrapod FSH and LH, respectively.     

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

Information on the pituitary gonadotropins in Chelonia mydasrepresents some of the most complete data available for anyreptile and thus provides an important basis for evaluatingevolutionary processes in tetrapod endocrine physiology. Thetwo gonadotropins isolated from Chelonia pituitary glands showclear chemical and immunological homologies to mammalian follicle-stimulatinghormone (FSH) and luteinizing hormone (LH). However, receptorstudies and biological tests indicate that the functions ofthese hormones may be different in turtles and mammals. In particular,Chelonia LH shows an unusual ability to interact with FSH receptorsites and to stimulate physiological functions normally attributedto FSH. Results with Chelonia LH demonstrate that errors mayarise from using mammalian hormones to investigate reproductionin turtles. Measurements of endogenous gonadotropin levels inthe plasma of breeding and nesting Chelonia provide a differentperspective of the potential roles of the FSH and LH from thatobtained in physiological tests. In particular, FSH and LH secretionare markedly dissociated during the nesting cycle; FSH has onlya transient peak during oviposition, whereas LH, along withprogesterone, displays a pronounced "ovulatory" surge in theday following nesting. Preliminary studies with synthetic gonadotropicreleasing factors in Chelonia suggest that these may be usefulin inducing reproductive changes.     

Immunohistochemical evidence that follicle-stimulating hormone and luteinizing hormone reside in separate cells in the chicken pituitary.

As is the case in other tetrapod species, the chicken gonadotropins LH and FSH consist of a common alpha subunit and a hormone-specific beta subunit. Gonadotrophs containing LH were shown earlier to be distributed throughout both the caudal and cephalic lobes of the chicken anterior pituitary, but the cellular distribution of FSH in avian species is still uncertain. The purpose of this study was to determine the cellular distribution of FSH-containing chicken gonadotrophs by use of FSH-specific monoclonal antibodies (mAbs). Three new mAbs toward chicken FSH were proven hormone specific by immunodetection of purified hormones on dot blots and by dual-label immunohistochemistry (IHC) on sagittal sections of chicken pituitaries. A rabbit antibody was used to detect chicken LH. Results showed that LH-containing gonadotrophs were densely distributed throughout the anterior pituitary, whereas gonadotrophs containing FSH were much less numerous; in addition, while also present in both lobes, FSH-positive cells were largely absent from the outer margin of the gland. Dual-label IHC revealed that LH and FSH reside almost exclusively in separate gonadotrophs. The identity of FSH-containing cells was further confirmed through use of an antibody to the chicken alpha subunit, which showed that FSH immunoreactivity was always colocalized with the alpha subunit. Our results suggest the possibility that production and secretion of LH and FSH may be regulated differently in chickens than in most other species studied to date.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

Puberty in the male pony: Plasma gonadotropin concentrations and the effects of castration

Ten male ponies were studied from 17 December 1978 through 9 August 1979. Six of the colts were born the previous spring (1978) and four were born during the previous summer. Three of the spring-born colts had been castrated at 4 months of age. Based on the presence of spermatozoa in the epididymis, all spring-born colts ($\text{3}\text{3}$), but only one summer-born colt ($\text{1}\text{4}$) had reached puberty by the end of the project (August). Spermatogenesis was significantly more advanced in the spring-born colts than in the summer-born colts.Concentrations of FSH and LH in the intact males remained constant from December through August, and levels were significantly lower than for the long-term castrated males throughout this period. In the long-term castrates, concentrations of FSH and LH increased from late winter and early spring to the highest levels during late spring and summer.On 9 August, the three spring-born colts (approximately 16 months of age) were castrated. The four summer-born colts (approximately 12–13 months old) were randomly assigned to either castrate (n=2) or hemicastrate (n=2) groups, and surgery was done on all colts on the same day. Both gonadotropins increased following castration in spring-born males. Concentrations of FSH and LH did not change following hemicastration.     

Effects of intraventricular brain injections of neurotransmitters on colonic temperature in morphine-tolerant rats

A sensitive and specific radioimmunoassay for LH-releasing hormone (LHRH) was developed. Using a specific antibody we have attempted to define or dissociate a separate FSHRH, antigenically distinct from LHRH. In an $\text{in vitro}$ system, LH release by hypothalamic extract was inhibited by a certain dose of LHRH antiserum but FSH release was not affected. Diurnal patterns of LHRH, FSH and prolactin were studied but no clear cyclic changes were shown. LHRH and LH levels in the serum were completely dissociated. We suggest that negative feedback systems play a more critical role than hypothalamic LHRH in the release of LH.     

Variability in gonadotrophin preparations as a factor in the superovulatory response

Much of the variability in superovulatory response has been attributed to variation in ovarian response of individual animals. Alternatively, differences in the relative abundance of FSH and LH activity in gonadotrophin preparations may contribute to superovulatory variation. This report presents evidence for variability in LH and FSH activity among equine chorionic gonadotrophin, porcine FSH and human menopausal gonadotrophins. Lower ratios of $\text{FSH}\text{LH}$ activity appeared to reduce ovulatory success in rats, and addition of PLH to FSH-P reduced superovulation in crossbred cows.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the $\text{in vitro}$ release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all $\text{in vitro}$ experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Effect of bromocriptine on the hypothalamo-pituitary function in adult male rats.

Daily oral administration of bromocriptine (50 μg/kg) to adult male rats, suppressed serum prolactin levels. The pituitary prolactin levels remained unaltered. Serum FSH levels as well as pituitary FSH levels showed no significant change as compared to the controls. Serum LH levels were significantly decreased in spite of the high pituitary LH levels, in bromocriptine treated rats. In the drug treated rats, $\text{in vitro}$ sensitivity of the pituitary to the exogenous LH-RH was not altered; whereas hypothalamic LH-RH content was considerably lowered. These observations suggest the possible effect of bromocriptine on the synthesis of LH-RH in the hypothalamus which leads to the accumulation of LH in the pituitary and decline of serum LH.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.     

Chronic treatment with valium (diazepam) fails to affect the reproductive system of the male rat

Male rats treated chronically with high doses of Valium (50mg/ Kg/day; 10 days) failed to exhibit changes in their reproductive system. Testicular and prostate weights, serum testosterone (T) and LH were unaffected. Testes and pituitary tissue stimulated $\text{in}$$\text{vitro}$ with LH and GnRH, respectively, released normal amounts of T, LH and FSH. Brain benzodiazepine receptors were slightly but significantly elevated by Valium treatment as well as by castration. We conclude that the male reproductive system is resistant to chronic Valium treatment even though the brain levels of benzodiazepine receptors are not.     

Evidence for an FSH-releasing factor in the posterior portion of the rat median eminence

To test the hypothesis that an FSH-releasing factor might be contained within the posterior portion of the median eminence (ME), the anterior half of the ME (aME) and the posterior half of the ME (pME) were removed separately from the brains of adult male rats and extracted in 0.2 N acetic acid. LH and FSH-releasing activities of the extracts were measured $\text{in}$$\text{vitro}$ by incubating 8 hemipituitaries from adult male rats for 6 h at a dose of 5 tissue equivalents and determining the radioimmunoassayable LH and FSH released into the medium. LH release induced by the aME extracts was significantly greater than that induced by the pME in both experiments, whereas there were no differences in FSH release between aME and pME extracts. A significant dose-related increase in FSH release was noted in this system when 1 and 2 ng of synthetic LHRH were tested which indicates that the assay was sensitive to different amounts of LHRH with regard to FSH-releasing action. The content of immunoreactive LHRH in the extracts was almost twice as high in the aME as in the pME. Therefore, the results indicate that the pME has greater FSH-releasing activity than can be accounted for by its content of LHRH. The additional FSH-releasing activity is presumably due to an FSH-releasing factor distinct from LHRH.     

The impact of dose of FSH (Folltropin) containing LH (Lutropin) on follicular development, estrus and ovulation responses in prepubertal gilts

FSH is favored over chorionic gonadotropins for induction of estrus in various species, yet little data are available for its effects on follicle development and fertility for use in pigs. For Experiment 1, prepubertal gilts (n = 36) received saline, 100 mg FSH, or FSH with 0.5 mg LH. Treatments were divided into six injections given every 8 h on Days 0 and 1. Proportions of gilts developing medium follicles were increased for FSH and FSH-LH (P < 0.05) compared to saline, but follicles were not sustained and fewer hormone-treated gilts developed large follicles (P < 0.05). No gilts expressed estrus and few ovulated. Experiment 2 tested FSH preparations with greater LH content. Prepubertal gilts (n = 56) received saline, FSH-hCG (100 mg FSH with 200 IU hCG), FSH-LH5 (FSH with 5 mg LH), FSH-LH10 (FSH with 10 mg LH), or FSH-LH20 (FSH with 20 mg LH). FSH-LH was administered as previously described, while 100 IU of hCG was given at 0 h and 24 h. Hormone treated gilts showed increased (P < 0.05) medium and large follicle development, estrus (>70%), ovulation (100%), and ovulation rate (>30 CL) compared to saline. There was an increase (P < 0.05) in the proportion of hormone-treated gilts with follicular cysts at Day 5, but these did not persist to Day 22. These gilts also showed an increase in poorly formed CL (P < 0.05). FSH alone or with small amounts of LH can induce medium follicle growth but greater amounts of LH at the same time is needed to sustain medium follicles, stimulate development of large follicles and induce estrus and ovulation in prepubertal gilts.     

Purification of equine gonadotropins and comparative study of their acid-dissociation and receptor-binding specificity

A method for the simultaneous purification of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from equine pituitaries is briefly described. Different forms of each hormone were obtained. The total yield of LH was 24.2 mg·kg^?1 with a recovery of 22% and the yield of FSH was 26 mg·kg^?1 with a recovery of 34%. The specific activities of both hormones, measured in homologous equine radio-receptor assays are equal to or higher than those of the preparations described so far. In all species studied so far the acid-dissociation curves of LH and FSH are similar; this is an agreement with the view that the binding of the common α-subunit and the specific β-subunits involves polypeptide regions which are identical in both hormones. In contrast, the acid-dissociation pK_a of equine LH was found to be considerably lower (3.9) than that of equine FSH (5.8). The equine gonadotropins exhibit a much lower specificity with receptors of a porcine testicular fraction compared with an equine fraction. Equine LH exhibited a binding activity on FSH receptors from a porcine testicular fraction equal to 20% that of equine FSH instead of only 1% for an equine binding fraction. Similarly, all the equine FSH preparations tested exhibited a five-fold higher binding-activity on porcine LH receptors than on equine LH receptors. In the porcine system, pregnant mare serum gonadotropin behaved like equine LH towards LH and FSH receptors. In contrast, on equine binding fraction, pregnant mare serum gonadotropin was only 4% as active as equine LH and was devoid of FSH activity. All the data we have obtained are consistent with the ‘negative specificity’ model we proposed recently.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused invitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused $\text{in}$$\text{vitro}$ were measured in order to compare PG changes in this model system with those that occur $\text{in}$$\text{vivo}$ and in isolated, LH-treated follicles $\text{in}$$\text{barvitro}$. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.