Changes in the content and distribution of microtubule associated protein 2 in the hippocampus of the rat during the estrous cycle

The molecular mechanisms involved in the regulation of synaptic plasticity in the hippocampus during the estrous cycle of the rat are not completely understood. Because this process implicates changes in neuronal cytoskeleton organization, we analyzed the content of microtubule associated protein 2 (MAP2) and Tau in the hippocampus and the frontal cortex of the rat by Western blot, as well as the hippocampal distribution of MAP2 during the estrous cycle by immunohistochemistry. In the hippocampus the lowest content of MAP2 was found on diestrus day, and it significantly increased at proestrus. This increase was maintained on estrus and metestrus days. In the frontal cortex MAP2 content did not significantly change during the estrous cycle. In contrast, the content of Tau did not vary during the estrous cycle in either the hippocampus or the frontal cortex. The immunohistochemical analysis showed an increase in dendrite thickness and in dendritic branching in the CA1 region on proestrus day, as well as an aggregation of MAP2 in apical dendrites near to pyramidal somata on this day in comparison with diestrus. We suggest that changes in the content and neuronal distribution of MAP2 are involved in the structural changes that occur in the hippocampus of the rat during the estrous cycle, and that these variations are related to changes in estradiol and progesterone levels.     

Sex differences,laterality, and hormonal regulation of androgen receptor immunoreactivity in rat hippocampus

Sex differences, laterality, and hormonal regulation of androgen receptor (AR) immunoreactivity in rat hippocampal CA1 pyramidal cells were examined using the PG21 antibody. Adult male rats were either castrated or sham-operated at least 2 weeks prior to sacrifice. Gonadally intact females were sacrificed on the day of proestrus. Animals received an injection of either testosterone propionate (TP) or vehicle 2 h prior to sacrifice. Within CA1, both the intensity of staining and the number of AR+ cells were assessed. AR immunostaining was detected in all the groups with marked variation among them. The overall ranking of staining intensity was: gonadally intact males > females given TP > castrated males given TP > females > castrated males given vehicle. The number of AR+cells within subregions of CA1 showed the same basic pattern: among control-treated animals, gonadally intact males have more than females, but castrated males have the least, and acute TP treatment increases the number in both sexes. The increased level of AR immunoreactivity in CA1 of castrated males following acute TP treatment suggests that testicular androgens in adulthood normally increase AR immunoreactivity there, producing a sex difference favoring males in gonadally intact animals. We also found a higher number of AR+ CA1 cells on the left than on the right, but only in gonadally intact males and in females given TP. These results suggest that a laterality of AR distribution in the rat hippocampus may lead to lateralities in hippocampal structure and function.     

Natural fluctuation and gonadal hormone regulation of astrocyte immunoreactivity in dentate gyrus

The number and the surface density of cells immunoreactive for the specific astrocytic marker glial fibrillary acidic protein (GFAP), were evaluated in both the hilus of the dentate gyrus and the granular layer of the vermis of the cerebellar cortex of adult female rats during the different phases of the estrous cycle, after ovariectomy and after the pharmacological administration of estradiol and/or progesterone to overiectomized rats. Although no significant differences were detected in the number of immunoreactive cells among the different experimental groups studied, their surface density showed significant changes in the hilus of the dentate gyrus. The surface density of immunoreactive cells was increased in the afternoon of proestrus and on the morning of estrus compared to the morning of proestrus, diestrus, and metestrus, was decreased after ovariectomy, and showed a dose-dependent increase in ovariectomized rats injected with 17β estradiol (1, 10, or 300 μg/rat), alone or in combination with progesterone (500 μg/rat). In contrast, it was not affected by the administration of 17β estradiol (300 μg/rat). The surface density of immunoreactive cells was significantly increased over control values by 5 h after the injection of 17β estradiol (300 μg/rat) and as early as 1 h after the administration of progesterone. The separate injection of either 17β estradiol or progesterone had smaller effects on the surface density of immunoreactive cells than did the administration of both hormones together. The surface density of GFAP-immunoreactive cells reached maximal values by 24 h after the administration of 17β estradiol and/or progesterone and returned to control levels by 48 h after the combined injection of progesterone and 17β estradiol, while in the rats that were injected with only one of the two hormones, the surface density of immunoreactive cells remained over control values for at least 9 days. No such hormonal effects on GFAP-immunoreactive cells were observed in the cerebellar cortex. © 1993 John Wiley & Sons, Inc.     

The Role of the δ GABA(A) Receptor in Ovarian Cycle-Linked Changes in Hippocampus-Dependent Learning and Memory

The δ subunit of the GABA_AR is highly expressed in the dentate gyrus of the hippocampus where it mediates a tonic extrasynaptic inhibitory current that is sensitive to neurosteroids. In female mice, the expression level of the δ subunit within the dentate gyrus is elevated in the diestrous relative to estrous phase of the estrous cycle. Previous work in our lab found that female δ-GABA_AR KO mice showed enhanced hippocampus-dependent trace but normal hippocampus-independent delay fear conditioning. Wild-type females in this study showed a wide range of freezing levels, whereas δ-GABA_AR KO mice expressed only high levels of fear. We hypothesized that the variability in the wild-type mice may have been due to estrous cycle-mediated changes in the expression of the δ-GABA_AR, with low levels of freezing in mice that were in the diestrous phase when dentate gyrus tonic inhibition is high. In the present study we tested this hypothesis by utilizing contextual, delay, and trace fear conditioning protocols in mice that were trained and tested in either the diestrous or estrous phases. Consistent with our hypothesis, we found a significant impairment of hippocampus-dependent learning and memory during diestrus relative to estrus in wild-type mice and this impairment was absent in δ-GABA_AR mice. These findings argue that the δ-GABA_AR plays an important role in estrous cycle-mediated fluctuations in hippocampus-dependent learning and memory.     

Amygdala activation by corticosterone alters visceral and somatic pain in cycling female rats

Irritable bowel syndrome (IBS) is often seen in women, and symptom severity is known to vary over the menstrual cycle. In addition, activation of the hypothalamic-pituitary-adrenal (HPA) axis enhances symptomology and patients with IBS have increased activation of the amygdala, a brain region known to facilitate HPA output. However, little is known about the effects of amygdala activation during different stages of the menstrual cycle. We therefore investigated the effects of amygdala activation on somatic and visceral pain perception over the rat estrous cycle. Female Wistar rats were implanted with either corticosterone (Cort) or cholesterol as a control onto the dorsal margin of the central amygdala. Visceral sensitivity was quantified by recording the visceromotor response (VMR) to colorectal distension (CRD) and somatic sensitivity was assessed via the Von Frey test. In cholesterol controls, both visceral and somatic sensitivity varied over the estrous cycle. Rats in proestrus/estrus responded to CRD with an increased VMR compared with rats in metestrus/diestrus. Somatic sensitivity followed a similar pattern with enhanced sensitivity during proestrus/estrus compared with metestrus/diestrus. Elevated amygdala Cort induced visceral hypersensitivity during metestrus/diestrus but had no effect during proestrus/estrus. In contrast, elevated amygdala Cort increased somatic sensitivity during both metestrus/diestrus and proestrus/estrous. These results suggests that amygdala activation by Cort eliminates spontaneously occurring differences in visceral and somatic pain perception, which could explain the lowered pain thresholds and higher incidence of somatic pain observed in women with IBS.     

Regional alterations in brain amino acids during the estrous cycle of the rat

Concentrations of 11 amino acids, including the neurotransmitters GABA, glutamate, aspartate, glycine and taurine, were determined in 12 brain regions of female rats during different stages of the estrous cycle. In addition, amino acids and sex hormone levels were determined in plasma. All sample collections were done in the forenoon between 9 and 11 a.m. Most regional amino acid levels measured did not change signficantly during estrous cycle, but significant alterations were found for GABA and glutamate in hypothalamus. Both amino acids were slightly decreased in hypothalamus during proestrus, which might reflect an alteration of GABA turnover in response to the high estrogen levels during this stage. A decreased glutamate level during proestrus was also found in thalamus, while both glutamate and GABA did not vary throughout estrous cycle in any of the other examined regions, including substantia nigra, amygdala, striatum, cortex and hippocampus. When diestrus was subdivided according to progesterone levels, high levels of this hormone seemed to be associated with effects on metabolism of certain amino acids, including glycine in substantia nigra, alanine in thalamus and threonine in pons/medulla. However, the few changes in regional amino acid concentrations found during the estrous cycle were so small that the functional significance of these changes cannot be ascertained without further determination of the cellular or subcellular compartments of brain tissue involved.     

Prolactin receptors in the rat mammary gland. Change during the estrous cycle

Ovine prolactin (o-PRL) binding to mammary gland membranes was studied during the estrous cycle in the rat. Groups of rats were decapitated throughout the 4-day estrous cycle at 10 h00 on the days of diestrus I, diestrus II and estrus and at 10 h00, 12 h00, 16 h00 during the day of proestrus. Daily vaginal smears were taken to determine the stage of the estrous cycle which was also controlled by PRL and LH serum levels. Prolactin receptors were quantified in the 100 000 g pellet. For one Scatchard analysis, mammary gland membranes from 5 animals were pooled. Results given are the mean of 4 or 5 pools. Results obtained showed that the apparent affinity constant (KA) remained unchanged during the days of diestrus II and at all the times studied of proestrus and showed a slight but significant decrease on the days of estrus and diestrus I (or metestrus). The binding capacity did not vary from the day of diestrus II to the proestrus 16h00 (11.3 +/- 2.8 fmoles/mg protein) but sharply increased on the day of estrus (190.4 +/- 35.9 fmoles/mg protein). Binding capacity remained elevated on the day of diestrus I. This increase of PRL receptors on the day of estrous would appear to be an important step in preparing mammary gland for pregnancy and lactation.     

Expression of ghrelin in the porcine hypothalamo-pituitary-ovary axis during the estrous cycle

Ghrelin, a 28-amino acid acylated peptide produced mainly by the stomach, has various functions. Recent studies focus on its endocrine and/or paracrine effects in the regulation of the hypothalamo-pituitary-gonadal axis, that is, the role in reproduction. Previous data have shown that variation of ghrelin depended on the phases of estrous cycle in adult rat ovary. This study was to investigate the expression of ghrelin in the cyclic porcine hypothalamo-pituitary-ovary axis and stomach by semiquantitative RT-PCR and immunohistochemical method. Twenty virginal gilts were classified into four groups as the proestrus, estrus, diestrus1 and diestrus2. Results showed that expression of ghrelin mRNA in the hypothalamus changed with the estrous cycle, i.e., with the highest level in the proestrus and the lowest in the estrus. In the pituitary, the pattern of ghrelin mRNA expression during estrous cycle markedly decreased in the estrus and diestrus1. In the ovary, ghrelin mRNA exhibited with the highest level in the diestrus2 and the lowest in the proestrus, which was different from those in the hypothalamus and pituitary. In the stomach, the expression of ghrelin mRNA had the same tendency as that of the porcine ovary. In immunohistochemical experiment, ghrelin immunoreactive cells were predominantly located in the luteal compartment and growing follicles in the luteal phase of ovary. However, only few ghrelin immunoreactive cells were found in the proestrus ovary. In gastric mucosa, ghrelin immunoreactive cells were detected in the estrus, diestrus1 and diestrus2, but few ghrelin positive cells were seen in the proestrus. Results suggest that ghrelin may play a major role in the endocrine network that integrates energy balance and reproduction.     

Alterations of Glial Cells in the Mouse Hippocampus During Postnatal Development

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.     

Sexual dimorphism in the content of progesterone and estrogen receptors, and their cofactors in the lung of adult rats

Progesterone and estradiol play an important role in the regulation of lung functions such as ventilation and vasoconstriction. The genomic actions of progesterone and estradiol are mediated by their nuclear receptors: progesterone receptors (PR) and estrogen receptors (ER). These actions are linked to interactions between steroid receptors and some cofactors that function as coactivators or corepressors. In this work we determined the content of PR isoforms, ER-beta, one coactivator (SRC-1), and one corepressor (SMRT) in the lung of both female rats during the estrous cycle and intact males by Western blot. The rat lung presented a higher content of PR-A than that of PR-B during the estrous cycle. The highest content of both PR isoforms was observed on the day of proestrus whereas the lowest one was found on the day of estrus. In contrast, the content of ER-beta was the lowest on the day of proestrus and it increased at estrus. The content of SRC-1 and SMRT increased on the day of diestrus. SRC-1 content decreased at proestrus and estrus, while SMRT content decreased at proestrus and increased again at estrus. In the lung of adult male rats the content of PR isoforms, ER-beta and SMRT was lower than in that of females, whereas the content of SRC-1 was similar in both sexes. Our results suggest a sexual dimorphism in the content of PR, ER-beta, and SMRT in the rat lung as well as a relation of their content to the physiological levels of progesterone and estradiol.     

Gender and estrous cycle influences on behavioral and neurochemical alterations in adult rats neonatally administered ketamine

Neonatal N‐methyl‐D‐aspartate (NMDA) receptor blockade in rodents triggers schizophrenia (SCZ)‐like alterations during adult life. SCZ is influenced by gender in age of onset, premorbid functioning, and course. Estrogen, the hormone potentially driving the gender differences in SCZ, is known to present neuroprotective effects such as regulate oxidative pathways and the expression of brain‐derived neurotrophic factor (BDNF). Thus, the aim of this study was to verify if differences in gender and/or estrous cycle phase during adulthood would influence the development of behavioral and neurochemical alterations in animals neonatally administered ketamine. The results showed that ketamine‐treated male (KT‐male) and female‐in‐diestrus (KTF‐diestrus, the low estrogen phase) presented significant deficits in prepulse inhibition of the startle reflex and spatial working memory, two behavioral SCZ endophenotypes. On the contrary, female ketamine‐treated rats during proestrus (KTF‐proestrus, the high estradiol phase) had no behavioral alterations. This correlated with an oxidative imbalance in the hippocampus (HC) of both male and KTF‐diestrus female rats, that is, decreased levels of GSH and increased levels of lipid peroxidation and nitrite. Similarly, BDNF was decreased in the KTF‐diestrus rats while no alterations were observed in KTF‐proestrus and male animals. The changes in the HC were in contrast to those in the prefrontal cortex in which only increased levels of nitrite in all groups studied were observed. Thus, there is a gender difference in the adult rat HC in response to ketamine neonatal administration, which is based on the estrous cycle. This is discussed in relation to neuropsychiatric conditions and in particular SCZ. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 519–532, 2016     

Chronological Distribution of Rip Immunoreactivity in the Gerbil Hippocampus During Normal Aging

Age-dependent studies on oligodendrocytes, which are the myelinating cells in the central nervous system, have been relatively less investigated. We examined age-dependent changes in Rip immunoreactivity and its protein level in the gerbil hippocampus during normal aging using immunohistochemistry and Western blot analysis with Rip antibody, an oligodendrocyte marker. Rip immunoreactivity and its protein level in the hippocampal CA1 region significantly increased at postnatal month 3 (PM 3). Thereafter, they decreased in the hippocampal CA1 region with age. At PM 24, Rip immunoreactive processes in the hippocampal CA1 region markedly decreased in the stratum radiatum. In the hippocampal CA2/3 region and dentate gyrus, the pattern of changes in Rip immunoreactivity and its protein level was similar to those in the hippocampal CA1 region; however, no significant changes were found in the CA2/3 region and dentate gyrus at various age stages. These results indicate that Rip immunoreactivity and protein level in the hippocampal CA1 region decreases significantly at PM 24 compared to the CA2/3 region and dentate gyrus.     

非基因型雌激素膜性受体GPR30在生后雌性大鼠海马内的发育学表达与亚细胞水平定位研究

为了研究非基因型雌激素膜性受体GPR30对海马的结构和功能的调节作用,应用硫酸镍铵增强显色的免疫组化技术以及酶标免疫电镜技术,观察了生后雌性大鼠海马内GPR30表达的变化及其免疫阳性产物在神经元亚细胞水平的定位情况．结果显示,GPR30免疫阳性产物主要位于海马CA区的锥体层神经元与齿状回颗粒层的神经元内,其表达水平随发育呈增加趋势．P0时在雌性大鼠海马未发现明显GPR30免疫阳性反应,P7后免疫阳性物质开始在CA2出现,P14时见于 CA1、CA2和齿状回,P30和P60主要见于CA1、CA2、CA3和齿状回．在光镜下,GPR30免疫阳性产物位于细胞核外的胞浆中,细胞核未见免疫阳性反应．在透射电镜下可见其位于神经元的胞浆内,可能主要是粗面内质网,也可见于线粒体和细胞膜．以上结果证实,GPR30是一种位于细胞核外的、非基因型作用的雌激素受体,可能参与了雌激素对海马锥体神经元突触可塑性和学习记忆等功能的调节,还可能参与了对齿状回成年神经干细胞某些活动的调节．     

小熊猫繁殖周期血清雌二醇和孕酮含量变化

为研究小熊猫繁殖周期血清雌二醇、孕酮含量变化规律,采用化学发光免疫分析法连续16 次测定了2只成体雌性小熊猫血清雌二醇和孕酮含量变化,历经发情间期、发情期和两次妊娠期;连续9次测定了7只小熊猫妊娠期的孕酮含量变化。结果:(1)发情间期,小熊猫血清雌二醇的水平一直维持在低水平(基础水平),进入发情前期,血清雌二醇水平明显升高,在发情期一直维持高水平,配种后迅速降至基础水平; (2)小熊猫血清孕酮含量在发情间期和发情期均维持在较低水平,直至发情期过后才出现升高,在妊娠期一直维持高水平,峰值出现在5 月;(3)发情的小熊猫不论妊娠与否,在妊娠期内血清孕酮含量均维持在高水平。研究表明:小熊猫血清雌二醇、孕酮含量变化能直接反映其繁殖规律,雌二醇对启动雌性小熊猫季节性繁殖起重要作用;在妊娠期内小熊猫血清孕酮含量升高不能作为判断小熊猫妊娠的标准;雌性小熊猫在妊娠期有假孕现象。     

Sex and hormonal cycle differences in rat brain levels of pain-related cannabimimetic lipid mediators

One important function of endocannabinoids and related lipid mediators in mammalian central nervous system is modulation of pain. Evidence obtained during the last decade shows that altered levels of these compounds in the brain accompany decreases in pain sensitivity. Such changes, if sexually dimorphic, could account for sex differences in pain and differences that occur during different phases of the hormonal cycle in females. To examine this possibility, we measured the levels of the pain-modulatory lipids anandamide, 2-arachidonoyl glycerol, N-arachidonoyl glycine, N-arachidonoyl gamma amino butyric acid, and N-arachidonoyl dopamine in seven different brain areas (pituitary, hypothalamus, thalamus, striatum, midbrain, hippocampus, and cerebellum) in male rats, and in female rats at five different points in the estrous cycle. The cerebellum did not demonstrate a change in endocannabinoid production across the estrous cycle, whereas all other areas tested showed significant differences in at least one of the compounds measured. These changes in levels occurred predominantly within the 36-h time period surrounding ovulation and behavioral estrus. Differences between males and females were measured as either estrous cycle-independent (all estrous cycles combined) or cycle-dependent (comparisons of males to each estrous cycle). In cycle-independent analyses, small sex differences were observed in the pituitary, hypothalamus, cerebellum, and striatum, whereas no differences were observed in the thalamus, midbrain, and hippocampus. In cycle-dependent analyses, the hypothalamus and pituitary showed largest sex differences followed by the striatum, midbrain, and hippocampus, whereas no sex differences were measured in thalamus and cerebellum. These data provide a basis for investigations into how differences in sex and hormonal status play a role in mechanisms regulating endocannabinoid production and pain.     

Spatial properties of astrocyte gap junction coupling in the rat hippocampus

Gap junction coupling enables astrocytes to form large networks. Its strength determines how easily a signalling molecule diffuses through the network and how far a locally initiated signal can spread. Changes of coupling strength are well-documented during development and in response to various stimuli. Precise quantification of coupling is needed for studying such modifications and their functional consequences. We therefore explored spatial properties of astrocyte coupling in a model simulating dye loading of single astrocytes. Dye spread into the astrocyte network could be characterized by a coupling length constant and coupling anisotropy. In experiments, the fluorescent marker Alexa Fluor 594 was used to measure these parameters in CA1 and dentate gyrus of the rat hippocampus. Coupling did not differ between regions but showed a temperature-dependence, partially owing to changes of intracellular diffusivity, detected by measuring coupling length constants but not the more variable cell counts of dye-coupled astrocytes. We further found that coupling is anisotropic depending on distance to the pyramidal cell layer, which correlated with regional differences of astrocyte morphology. This demonstrates that applying these new analytical approaches provides useful quantitative information on gap junction coupling and its heterogeneity.     

Striatal D-2 dopamine agonist binding sites fluctuate during the rat estrous cycle

Striatal D-2 dopamine (DA) antagonist and agonist binding sites were measured during the rat estrous cycle and compared to ovariectomized (OVX) rats. Dopaminergic D-2 antagonist binding sites were constant during the estrous cycle while agonist binding sites show a rapid and significant decrease of the ratio of high to low D-2 agonist binding sites from proestrus AM (PAM) to diestrus 1 (D1) and return to OVX value in diestrus 2 (DII). Thus, physiological fluctuations of hormones as occur during the estrous cycle can modulate extrahypothalamic biogenic amine activity, namely striatal DA systems which are not involved in the control of hormone secretion.     

Neuroendocrine disruption is associated to infertility in chronically stressed female rats

Infertility is a growing worldwide public health problem, and stress is a main factor exerting detrimental effects on female reproduction. However, knowledge regarding the neuroendocrine changes caused by chronic stress in females is limited. Therefore, this study assessed the effects of stress on hormones that control female reproduction during the proestrus and diestrus stages of the estrous cycle, as well as its effects on fertility. Adult females were assigned to either a control or a stress group. Stress consisted of exposure, for 15 min, to cold-water immersion daily for 30 days. Estrous cyclicity, female sexual behavior, as well as hypothalamic kisspeptin, gonadotropin releasing hormone (GnRH) content, serum luteinizing hormone (LH), estradiol (E₂), progesterone (P₄), corticosterone (CORT) and fertility were assessed after chronic stress. The results show that chronically stressed females exhibited disrupted estrous cyclicity, decreased receptivity, low pregnancy rates and lower numbers of fetuses. The content of Kisspeptin and GnRH in the Anteroventral Periventricular/medial Preoptic Area decreased during proestrus, while Kisspeptin increased in the Arcuate nucleus in proestrus and diestrus. Serum LH decreased only during proestrus, whereas E₂ and P₄ concentrations decreased during proestrus and diestrus, with a concomitant increase in CORT levels in both stages. As a whole, these results indicate that chronic stress decreases Kisspeptin content in AVPV nucleus and GnRH in POA in females, and might induce disruption of the LH surge, consequently disrupting estrous cyclicity and fertility, leading to lower rates of pregnancy and number of fetuses.