Kinesins are essential for the proper function of many types of polar cells, including epithelial cells, neurons, and sperm. Spermatogenesis is closely associated with many different kinesins. These kinesins participate in several fundamental processes, including mitotic and meiotic division, essential organelle transport, and the biogenesis of peculiar structures for the formation of mature sperm. Kinesin-13, kinesin-8, and the chromokinesin families cooperate to ensure normal sister chromatid congression and segregation. The kinesin-8 family motor KIF18A, kinesin-12 motors PAKRP/kinesin12A and PAKRP1L/kinesin12B, and other kinesin-like motors are essential in the process of homologous chromosome pairing and in the separation to create haploid gametes. During spermiogenesis, the responsibility of a handful of kinesin members lies in the maturation of spermatids into mature, motile, and intact spermatozoa. Such roles are completed upon the release of viable and functional sperm into the lumen of seminiferous tubules. In this process, KIFC1, KIF5C, KRP3A, and KRP3B may be involved in acrosome biogenesis; KIFC1, KIFC5, CHO2, KIF17b, and KIF3A probably contribute to nuclear shaping; KIF17b, KIF3A, and KLC3 are implicated in the tail formation process; and KIF20 and KRP3 likely participate in sperm translocation. KIF17b also exhibited postmeiosis transcriptional activities that are critical for the dramatic alterations observed in nuclear and cytoplasmic structures. This review summarizes the roles of kinesins during mitosis, meiosis, and spermiogenesis, and proposes several important issues for further investigation.

Generation of nonhuman primate models of human disease conditions will foster the development of novel therapeutic strategies. Callithrix jacchus, or the common marmoset, is a New World, nonhuman primate species that exhibits great reproductive fitness in captivity with an ovarian cycle that can be easily managed with pharmacological agents. This characteristic, among others, provides an opportunity to employ assisted reproductive technologies to generate embryos that can be genetically manipulated to create a variety of nonhuman primate models for human disease. Here, we review methods to synchronize the marmoset ovarian cycle and stimulate oocyte donors, and compare various protocols for in vitro production of embryos. In light of advances in genomic editing, recent approaches used to generate transgenic or genetically edited embryos in the marmoset and also future perspective are reviewed.

Summary Sentence

This review presents a summary of assisted reproductive technologies utilized to generate in vitro produced embryos in the common marmoset and describes prospects for genomic editing and transgenesis to create nonhuman primate models of human disease.

Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the presence of Gap26 that targets GJA1 (Cx43) and GJA4 (Cx37). Furtherwork showed that HCs from blastocysts produced after oocyte maturation in the presence of serum were open shortly after vitrification/warming, and this was prevented by Gap26. Gap26, applied for the exposure times used, inhibited Cx43 and Cx37 HCs while it did not have an effect on GJs. Interestingly, Gap26 had no effect on blastocyst degeneration or cell death. We conclude that blocking HCs protects embryos during vitrification and warming by a functional effect not linked to cell death.

Summary Sentence

Peptide targeting of connexins protects bovine blastocysts against vitrification/warming stress by preventing the opening of hemichannels that function as a leakage pathway.

To date, several groups have generated homologous models of endometriosis through the implantation of endometrial tissue fluorescently labeled by green fluorescent protein (GFP) or tissue from luciferase-expressing transgenic mice into recipient animals, enabling noninvasive monitoring of lesion signal. These models present an advantage over endpoint models, but some limitations persist; use of transgenic mice is laborious and expensive, and GFP presents poor tissue penetration due to the relatively short emission wavelength. For this reason, a homologous mouse model of endometriosis that allows in vivo monitoring of generated lesions over time and mimics human lesions in recipient mice would be most desirable.

In this regard, using C57BL/6 and B6N-Tyr^c-Brd/BrdCrCrl mice, we optimized a decidualization protocol to obtain large volumes of decidual endometrium and mimic human lesions. Subsequently, to obtain a more robust and reliable noninvasive monitoring of lesions, we used the fluorescent reporter mCherry, which presents deeper tissue penetration and higher photostability, showing that endometrial tissue was properly labeled with 1 × 10⁸ PFU/mL mCherry adenoviral vectors. mCherry-labeled endometriotic tissue was implanted in recipient mice, generating lesions that displayed characteristics typical of human endometriotic lesions, such as epithelial cells forming glands, local inflammation, collagen deposits, and new vessel formation. In vivo monitoring demonstrated that subcutaneous implantation on ventral abdomen of recipient mice provided the most intense and reliable signal for noninvasive lesion monitoring over a period of at least 20 days. This homologous model improves upon previously reported models of endometriosis and provides opportunities to study mechanism underlying endometriotic lesion growth and progression.

Summary Sentence

We created a cost-effective but accurate homologous mouse model of endometriosis that allows the study of growth and progression of endometriotic lesions over early time points in lesion development through noninvasive monitoring.

The precise timing of progesterone signaling through its cognate receptor, the progesterone receptor (PGR), is critical for the establishment and maintenance of pregnancy. Loss of PGR expression in the murine uterine epithelium during the preimplantation period is a marker for uterine receptivity and embryo attachment. We hypothesized that the decrease in progesterone receptor A (PGRA) expression is necessary for successful embryo implantation. To test this hypothesis, a mouse model constitutively expressing PGRA (mPgrA^LsL/+) was generated. Expression of PGRA in all uterine compartments (Pgr^cre) or uterine epithelium (Wnt7a^cre) resulted in infertility with defects in embryo attachment and stromal decidualization. Expression of critical PGRA target genes, indian hedgehog, and amphiregulin (Areg), wasmaintained through the window of receptivity while the estrogen receptor target gene, the leukemia inhibitory factor (Lif), a key regulator of embryo receptivity, was decreased. Transcriptomic and cistromic analyses of the mouse uterus at day 4.5 of pregnancy identified an altered group of genes regulating molecular transport in the control of fluid and ion levels within the uterine interstitial space. Additionally, LIF and its cognate receptor, the leukemia inhibitory factor receptor (LIFR), exhibited PGR-binding events in regions upstream of the transcriptional start sites, suggesting PGRA is inhibiting transcription at these loci. Therefore, downregulation of the PGRA isoform at the window of receptivity is necessary for the attenuation of hedgehog signaling, transcriptional activation of LIF signaling, and modulation of solutes and fluid, producing a receptive environment for the attaching embryo.

Summary Sentence

Expression of PGRA at the window of receptivity transcriptionally represses LIF signaling and aberrantly regulates hedgehog and solute signaling rendering the uterus unreceptive to the implanting embryo.

Factors delivered to offspring in colostrum within 2 days of birth support neonatal porcine uterine development. The uterine mRNA transcriptome is affected by age and nursing during this period. Whether uterine microRNA (miRNA) expression is affected similarly is unknown. Objectives were to (1) determine effects of age and nursing on porcine uterine miRNA expression between birth and postnatal day (PND) 2 using miRNA sequencing (miRNAseq) and; (2) define affected miRNA–mRNA interactions and associated biological processes using integrated target prediction analysis.At birth (PND 0), gilts were euthanized, nursed ad libitum, or gavage-fed milk replacer for 48 h. Uteri were collected at birth or 50 h postnatal. MicroRNAseq data were validated using quantitative real-time PCR. Targets were predicted using an established RNA database generated from the same tissues. For PND 2 versus PND 0 comparisons, 31 differentially expressed (DE) miRNAs were identified for nursed, and 42 DE miRNAs were identified for replacer-fed gilts. Six DE miRNAs were identified for nursed versus replacer-fed gilts on PND 2. Target prediction for inversely correlated DE miRNA–mRNA pairings indicated 20 miRNAs targeting 251 mRNAs in nursed, versus 29 miRNAs targeting 585 mRNAs in replacer-fed gilts for PND 2 versus PND 0 comparisons, and 5 miRNAs targeting 81 mRNAs for nursed versus replacer-fed gilts on PND 2. Biological processes predicted to be affected by age and nursing included cell-to-cell signaling, cell morphology, and tissue morphology. Results indicate novel age- and lactocrine-sensitive miRNA–mRNA relationships associated with porcine neonatal uterine development between birth and PND 2.

Summary Sentence

Comprehensive microRNA–mRNA analyses identified novel age- and lactocrine-sensitive porcine uterine microRNAs, microRNA–mRNA interactions, and biological processes associated with porcine neonatal uterine development.

Embryonic poly(A)-binding protein (EPAB)-deficient mice are infertile due to defects in both the oocyte and the somatic cells of the ovary. Since EPAB is oocyte specific, the abnormalities in the somatic compartment of Epab^-/- mice are likely due to factors inherent to the oocyte. Herein, we investigated whether oocyte—somatic communication is disrupted as a result of EPAB deficiency. We found that gap junctions are disrupted at the late preantral stage of folliculogenesis in Epab^-/- mice and remain disrupted in cumulus-enclosed oocytes (COCs) from antral follicles. Consistent with the timing of gap junction dysfunction, F-actin staining of transzonal processes (TZPs) is lower in Epab^-/- follicles at the late preantral stage and completely absent in Epab^-/- COCs. Epab^-/- oocytes express significantly lower levels of the junction protein E-cadherin, which is likely to be a contributing factor leading to premature TZP retraction. Overall, these results demonstrate that EPAB is important for oocyte—somatic communication by maintaining TZPs and gap junctions at the preantral stage of folliculogenesis.

Summary Sentence

EPAB is an oocyte-specific translational regulator that has a precise role at the preantral stage of follicle development for establishing communication between the oocyte and somatic cells.

Formation of complexes between soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins on opposing membranes is the minimal requirement for intracellular membrane fusion. The SNARE, syntaxin 2, is found on the sperm plasma membrane and a second SNARE, vesicle associated membrane protein 2 (VAMP2, also known as synaptobrevin 2, SYB2), is on the apposing outer acrosomal membrane. During the acrosome reaction, the outer acrosomal membrane fuses at hundreds of points with the plasma membrane. We hypothesized that syntaxin 2 and VAMP2 redistribute within their respective membranes prior to the acrosome reaction to form trans-SNARE complexes and promote membrane fusion. Immunofluorescence and superresolution structured illumination microscopy were used to localize syntaxin 2 and VAMP2 in mouse sperm during capacitation. Initially, syntaxin 2 was found in puncta throughout the acrosomal region. At 60 and 120 min of capacitation, syntaxin 2 was localized in puncta primarily in the apical ridge. Although deletion of bicarbonate during incubation had no effect, syntaxin 2 puncta were relocated in the restricted region in less than 20% of sperm incubated without albumin. In contrast, VAMP2 was already found in puncta within the apical ridge prior to capacitation. The puncta containing syntaxin 2 and VAMP2 did not precisely co-localize at 0 or 60 min of capacitation time. In summary, syntaxin 2 shifted its location to the apical ridge on the plasma membrane during capacitation in an albumin-dependent manner but VAMP2 was already localized to the apical ridge. Puncta containing VAMP2 did not co-localize with those containing syntaxin 2 during capacitation; therefore, formation of trans-SNARE complexes containing these SNAREs does not occur until after capacitation, immediately prior to acrosomal exocytosis.

Summary Sentence

Although the sperm plasma membrane SNARE protein syntaxin 2 moves laterally, trans-SNARE complexes are not formed during capacitation.

While spermatozoa undergo epididymal maturation, they remain quiescent thanks to the establishment of a low luminal pH. This study is aimed at determining how epithelial cells lining the epididymal lumen work together tomaintain and regulate this acidicmilieu. In particular, we examined the relative contribution of clear cells (CCs) and principal cells (PCs) to this process. Functional analysis in the mouse cauda epididymidis (Cd) perfused in vivo showed that the pH of a control solution remained constant at pH 6.6 after perfusion through the Cd lumen. In contrast, the pH of both an acidic (pH 5.8) and alkaline (pH 7.8) perfusate was progressively restored toward the control acidic pH. Pharmacological studies indicated the contribution of cystic fibrosis transmembrane regulator, previously shown to be present in the apical membrane of PCs, to the recovery from an acidic pH of 5.8. In addition, we found that CCs and PCs equally contribute to the recovery from an alkaline of 7.8, via the H⁺ pumping vacuolar ATPase (V-ATPase) located in CCs, and the Na⁺/H⁺ exchanger type 3 (NHE3) located in PCs. Immunofluorescence labeling showed apical membrane accumulation of the V-ATPase in CCs at pH 7.8, and its internalization at pH 5.8 compared to pH 6.6. Immunofluorescence showed expression of NHE3, but absence of NHE2, in PCs located in the Cd. RT-PCR and western blotting showed expression of NHE3 in all epididymal regions. Luminal 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (cpt-cAMP) partially inhibited luminal pH recovery from pH 7.8. However, cpt-cAMP induced an increase in V-ATPase apical membrane accumulation at this pH. Cell fractionation studies showed the apical accumulation of NHE3 from intracellular vesicles at pH 7.8 versus 6.6, and prevention of this effect by cpt-cAMP. These results indicate the participation of both CCs and PCs in the regulation of luminal pH in the epididymis. Our study also shows the dual role of PCs in and H⁺ secretion, and that this switch from base to acid secretion depends on the luminal environment. Characterization of the respective roles of CCs and PCs in the regulation of the optimal luminal condition for epididymal sperm maturation should provide new frameworks for the evaluation and treatment of male infertility.

Summary Sentence

This study shows the equal contribution of clear and principal cells to proton secretion, and the dual role of principal cells in bicarbonate and proton secretion, depending on luminal cues.

Here we report that the Kiss1 hexadecapeptide (Kiss1-16) directly regulates the functional form of gonadotropin-releasing hormone (GnRH) in the preoptic area (POA) of a scombroid fish model. In this study, we analyzed the localization of two kisspeptin (kiss1 and kiss2) neurons and two kisspeptin receptors (kissr1 and kissr2) in the brain of adult chub mackerel using in situ hybridization to determine whether the kisspeptin receptors co-localize with GnRH1 neurons. The kiss1-and kiss2-expressing neurons were mainly localized in the nucleus recessus lateralis (NRL) and the nucleus of the posterior recess (NRP) in the hypothalamus. Kissr1 was present in the anterior POA and the habenular nucleus. Kissr2 was widely distributed, including in the POA, lateral tuberal nucleus, NRL, and NRP. Notably, GnRH1 was expressed in neurons in the POA, and these neurons co-expressed kissr1. In contrast, kissr2 was expressed abundantly in the vicinity of GnRH1 neurons, but their co-expression did not seem to occur. We also characterized the endogenous mature form of the Kiss1 peptide. An in vitro reporter gene assay clearly showed that Kiss1-16 (HQDMSSYNFNSFGLRY-NH₂) was more potent at receptor activation than Kiss1 pentadecapeptide (Kiss1-15), which is the form of Kiss1 found in other fish species. This study strongly suggests that kisspeptin signaling, especially Kiss1 signaling, is important for regulating reproduction in scombroid fish.

Summary Sentence

Kiss1 hexadecapeptide (Kiss1-16) is the mature Kiss1 form in scombroid fish, chub mackerel, and may directly regulate reproductive axis via control of GnRH1 neuronal activity.

Angiogenesis in the ovary occurs rapidly as the ovarian follicle transforms into a mature corpus luteum. Granulosa cells produce vascular endothelial growth factor A (VEGFA) in response to the ovulatory gonadotropin surge. VEGFA is established as a key mediator of angiogenesis in the primate ovulatory follicle. To determine if additional VEGF family members may be involved in angiogenesis within the ovulatory follicle, cynomolgus monkeys (Macaca fascicularis) received gonadotropins to stimulate multiple follicular development, and human chorionic gonadotropin (hCG) substituted for the luteinizing hormone surge to initiate ovulatory events. Granulosa cells of monkey ovulatory follicles contained mRNA and protein for VEGFC and VEGFD before and after hCG administration. VEGFC and VEGFD were detected in monkey follicular fluid and granulosa cell-conditioned culture media, suggesting that granulosa cells of ovulatory follicles secrete both VEGFC and VEGFD. To determine if these VEGF family members can stimulate angiogenic events, monkey ovarian microvascular endothelial cells (mOMECs) were obtained from monkey ovulatory follicles and treated in vitrowith VEGFC and VEGFD. Angiogenic events aremediated via three VEGF receptors; mOMECs express all three VEGF receptors in vivo and in vitro. Exposure of mOMECs to VEGFC increased phosphorylation of AKT, while VEGFD treatment increased phosphorylation of both AKT and CREB. VEGFC and VEGFD increased mOMEC migration and the formation of endothelial cell sprouts in vitro. However, only VEGFD increased mOMEC proliferation. These findings suggest that VEGFC and VEGFD may work in conjunction with VEGFA to stimulate early events in angiogenesis of the primate ovulatory follicle.

Summary Sentence

VEGFC and VEGFD produced by granulosa cells of the ovulatory folliclemay contribute to follicular angiogenesis and ovulation.

In the zebrafish, no sex-determining gene has been identified, while some sex-related genes, such as cyp19a1a and amh, show sexually dimorphic expression. Interestingly, most of these genes are expressed in the somatic cells. With increasing evidence suggesting roles of germ cells in gonadal differentiation, there is an increasing interest in the factors released by the germ cells for the bidirectional communication between the two compartments. We have reported that Gdf9/gdf9 is an oocyte-specific factor in the zebrafish, similar to that of mammals. Whether and how Gdf9 is involved in gonadal differentiation is unknown. In this study, we compared the expression levels of gdf9, cyp19a1a, and amh among several other sex-related genes in the gonads before, during, and after sex differentiation. The expression of gdf9 started in the gonads before sex differentiation, and its level surged in the differentiated ovary. Its expression pattern was similar to that of cyp19a1a, but reciprocal to amh expression. Using recombinant zebrafish Gdf9 (rzfGdf9), we further showed that Gdf9 significantly suppressed the expression of amh while increased that of activin beta subunits (inhbaa and inhbb) in vitro. Although gdf9 and cyp19a1a showed co-expression during gonadal differentiation, we only observed a slight but not significant response of cyp19a1a to rzfGdf9. Knocking down the expression of gdf9 and cyp19a1a with vivo-morpholinos caused a male-skewed sex ratio. Our data suggested that Gdf9 is likely involved in promoting oocyte/ovary differentiation in the zebrafish and it may act by suppressing amh expression, at least partly, in the somatic cells.

Summary Sentence

Oocytes may influence gonadal differentiation by releasing growth differentiation factor 9.

Equine pregnancy is characterized by very high circulating concentrations of estrogens. The physiological roles of estrogens during equine gestation are largely unknown, although some studies suggest a role in the regulation of uterine artery hemodynamics and a relationship between low circulating estrogen concentrations and late pregnancy loss. The objectives of this experiment were to evaluate the effects of estrogen suppression on uterine artery hemodynamics and on pregnancy outcome. Estrogen synthesis was suppressed using letrozole, a potent aromatase inhibitor. Twelve pregnant mares were randomly assigned to a control (n = 6) or treatment (n = 6; 500 mg letrozole orally every 4 days) group with treatment starting at 240 days of gestation and continuing until parturition. Weekly serum samples were analyzed to determine testosterone, dehydroepiandrosterone sulfate, estradiol, estrone sulfate, progestins, and prostaglandin F2α metabolite concentrations. Ultrasonographic examinations were performed biweekly andmeasurements included uterine artery hemodynamics (diameter, pulsatility, and resistance indices), fetal growth using the diameter of the fetal eye, and placental evaluation using the combined thickness of the uterus and placenta. At parturition, gestational length, foal weight, and neonatal viability were determined. Letrozole suppressed estrogen synthesis during gestation by approximately 90% compared to control values. This large reduction in circulating estrogens had no effect on uterine artery hemodynamics, normal placental development, maintenance of pregnancy, or neonatal viability. However, neonates from letrozole-treated mares had lower (P < 0.05) birth weights than controls, suggesting that estrogens may play a role in fetal growth that is not mediated through regulation of uterine blood flow.

Summary Sentence

Chronic suppression of estrogen synthesis during the last trimester of equine gestation reduced fetal growth but had no effect on uterine artery hemodynamics, maintenance of pregnancy, or neonatal viability.

Chronic hypoxia during gestation suppresses large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel function and impedes uterine arterial adaptation to pregnancy. This study tested the hypothesis that chronic hypoxia has a direct effect in upregulating DNA methyltransferase (DNMT) and epigenetically repressing BK_Ca channel beta-1 subunit (KCNMB1) expression in uterine arteries. Resistance-sized uterine arteries were isolated from near-term pregnant sheep maintained at ∼300 m above sea level or animals acclimatized to high-altitude (3,801 m) hypoxia for 110 days during gestation. For ex vivo hypoxia treatment, uterine arteries from normoxic animals were treated with 21.0% O₂ or 10.5% O₂ for 48 h. High-altitude hypoxia significantly upregulated DNMT3b expression and enzyme activity in uterine arteries. Similarly, ex vivo hypoxia treatment upregulated DNMT3b expression and enzyme activity that was blocked by a DNMT inhibitor 5-aza-2′-deoxycytidine (5- Aza). Of importance, 5-Aza inhibited hypoxia-induced hypermethylation of specificity protein (SP) 1 binding site at the KCNMB1 promoter and restored transcription factor binding to the KCNMB1 promoter, resulting in the recovery of KCNMB1 gene expression in uterine arteries. Furthermore, 5-Aza blocked the effect of hypoxia and rescued BK_Ca channel activity and reversed hypoxia-induced decrease in BK_Ca channel-mediated relaxations and increase in myogenic tone of uterine arteries. Collectively, these results suggest that chronic hypoxia during gestation upregulates DNMT expression and activity, resulting in hypermethylation and repression of KCNMB1 gene and BK_Ca channel function, impeding uterine arterial adaptation to pregnancy.

Summary Sentence

Gestational hypoxia promotes hypermethylation and repression of KCNMB1 gene and BK_Ca channel function in uterine arteries by upregulating DNMTexpression and activity, leading tomaladaptation of uterine circulation during pregnancy.

Gestational diabetesmellitus (GDM) is a common obstetric complication. Half of women who have GDM will go on to develop type 2 diabetes. Understanding the mechanisms by which this occurs requires an animal model of GDM without ongoing diabetes at conception. C57Bl/6J mice react acutely to a high-fat, high-sucrose (HFHS) challenge. Here, we hypothesized that a periconceptional HFHS challenge will induce glucose intolerance during gestation. C57Bl/6J femalemice were placed on an HFHS either 1 or 3 weeks prior to mating and throughout pregnancy. Intraperitoneal glucose tolerance tests, insulin measurements, and histological analysis of pancreatic islets were used to assess the impact of acute HFHS. C57Bl/6J females fed HFHS beginning 1 week prior to pregnancy became severely glucose intolerant, with reduced insulin response to glucose, and decreased pancreatic islet expansion during pregnancy compared to control mice. These GDM characteristics did not occur when the HFHS diet was started 3 weeks prior to mating, suggesting the importance of acute metabolic stress. Additionally, HFHS feeding resulted in only mild insulin resistance in nonpregnant females. When the diet was discontinued at parturition, symptoms resolved within 3 weeks. However, mice that experienced glucose intolerance in pregnancy became glucose intolerant more readily in response to a HFHS challenge later in life than congenic females that experienced a normal pregnancy, or that were fed the same diet outside of pregnancy. Thus, acute HFHS challenge in C57Bl/6 mice results in a novel, nonobese, animal model that recapitulates the long-term risk of developing type 2 diabetes following GDM.

Summary Sentence

Exposure to a high-fat, high-sucrose diet just before and during pregnancy in mice impairs islet cell expansion, resulting in glucose intolerance of pregnancy and the development of glucose intolerance later in life.

Cold storage of the cauda epididymis, a male reproductive organ, is an efficient means of transporting genetically modified mice, an alternative to live animal shipment. The fertility of cold-stored sperm decreases in a time-dependent manner. However, the cause of the reduction in the fertility of sperm after cold storage remains unclear. It is known that cholesterol efflux from the sperm membrane is a trigger of capacitation. The dysfunction of cholesterol efflux due to changes in the membrane state in low temperatures may explain the reduced fertility. In this study, we examined the fertility (fertilization rate, acrosome reaction, and motility) and the amount of cholesterol and membrane fluidity in cold-stored sperm after treatment with two cholesterol acceptors, bovine serum albumin (BSA), and methyl-beta-cyclodextrin (MBCD). MBCD-treated sperm exhibited the highest rate of fertilization. Two-cell embryos derived from in vitro fertilization using 0.75-mM-MBCD-treated sperm after cold storage for 72 h developed to offspring. MBCD also displayed greater ability to remove cholesterol from the sperm membrane than BSA. The percentage of viable sperm with membrane destabilization was increased by MBCD in cold-stored sperm. The acrosome reaction strongly occurred in MBCD-treated sperm. In motility analysis, MBCD improved the lateral amplitude of head movement and beat frequency. These results suggest that MBCD-induced cholesterol efflux enhances membrane fluidity and promotes acrosome reaction and hyperactivation, resulting in improved fertility of cold-stored sperm.

Summary Sentence

Methyl-β-cyclodextrin removed cholesterol from the entire plasma membrane in cold-stored sperm and restored fertility by inducing the acrosome reaction and hyperactivation.

Interaction of Na/K-ATPase with its ligand ouabain has been implicated in the regulation of various biological processes. The objective was to investigate roles of Na/K-ATPase isoforms in formation and function of junctional complexes in Sertoli cells. Primary cultures of Sertoli cells were obtained by enzymatic digestion of 20-day-old rat testes and grown on Matrigel-coated dishes for 7 days. Sertoli cells predominantly expressed the ubiquitous isoform of Na/K-ATPase (ATP1A1), confirmed by immunoblotting, PCR, immunofluorescence, and mass spectrometry. Treatment of Sertoli cells with 50 nM ouabain increased transepithelial electrical resistance (TER) and expression of claudin 11 (tight junctions) and connexin 43 (gap junctions), whereas 1 mM ouabain had opposite effects. Involvement of Src-EGFR-ERK1/2-CREB pathway in ouabain-mediated expression of claudin 11 and connexin 43 was evaluated. Incubation of Sertoli cells with 50 nM ouabain increased content of p-Src, p-EGFR, p-ERK1/2, and p-CREB; in contrast, 1 mM ouabain decreased phosphorylation of these signalingmolecules. Preincubation of Sertoli cells with inhibitors of Src andMAPK pathways inhibited ouabain-induced effects on these signaling molecules, TER, and expression of claudin 11 and connexin 43. In conclusion, we inferred that ATP1A1 regulated Sertoli cell tight junctions and gap junctions through the Src-EGFR-ERK1/2-CREB pathway. Ouabain is an endogenous steroid; therefore, its interaction with ATP1A1 may be a critical signaling mechanism for the regulation of Sertoli cell function and male fertility.

Summary Sentence

The ATP1A1-Src-EGFR-ERK1/2-CREB pathway regulates expression of connexin 43 and claudin 11 in rat Sertoli cells; consequently, it is involved in formation and function of junctional complexes.

Cell junctions are necessary for spermatogenesis, and there are numerous types of junctions in testis, such as blood–testis barrier, intercellular bridge, and ectoplasmic specialization (ES). The details of their functions and construction are still unknown. To identify a novel protein essential to the function of a cell junction, we enriched testis membrane protein and analyzed it using a proteomics approach. Here, we report a novel ES protein, which is encoded on the X chromosome and an ortholog of hypothetical human protein KIAA1210. KIAA1210 is expressed in testis predominantly, localized to the sex body in spermatocyte, acrosome, and near ES. Moreover, KIAA1210 possesses a topoisomerase 2 (TOP2)-associated protein PAT1 domain, a herpes simplex virus 1 (HSV-1) large tegument protein UL36 hypothetical domain, and a provisional DNA translocase FtsK domain. Using IP-proteomics with specific antibody to KIAA1210, we identified proteins including TOP2 isoforms as components of a complex with KIAA1210, in cell junctions in testis. The interaction between KIAA1210 and TOP2 was confirmed by two different proteomic analyses. Furthermore, immunofluorescence showed that KIAA1210 and TOP2B co-localize around the sex body in spermatocyte, apical ES, and residual bodies in elongated spermatids. Our findings suggest that KIAA1210 may be essential cell junction protein that interacts with TOP2B to regulate the dynamic change of chromatin structures during spermiogenesis.

Summary Sentence

KIAA1210, which is a novel X-chromosome-linked protein, is localized to the acrosome and associates with ectoplasmic specialization, and has a direct or indirect interaction with DNA topoisomerase 2.

Mechanisms underlying obesity-associated reproductive impairment are ill defined. Hyperinsulinemia is a metabolic perturbation often observed in obese subjects. Insulin activates phosphatidylinositol 3-kinase (PI3K) signaling, which regulates ovarian folliculogenesis, steroidogenesis, and xenobiotic metabolism. The impact of progressive obesity on ovarian genes encoding mRNA involved in insulin-mediated PI3K signaling and xenobiotic biotransformation [insulin receptor (Insr), insulin receptor substrate 1 (Irs1), 2 (Irs2), and 3 (Irs3); kit ligand (Kitlg), stem cell growth factor receptor (Kit), protein kinase B (AKT) alpha (Akt1), beta (Akt2), forkhead transcription factor (FOXO) subfamily 1 (Foxo1), and subfamily 3 (Foxo3a), microsomal epoxide hydrolase (Ephx1), cytochrome P450 family 2, subfamily E, polypeptide 1 (Cyp2e1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1)] was determined in normal wild-type nonagouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 weeks of age. At 6 weeks, ovaries from obese mice had increased (P < 0.05) Insr and Irs3 but decreased (P < 0.05) Kitlg, Foxo1, and Cyp2e1 mRNA levels. Interestingly, at 12 weeks, an increase (P < 0.05) in Kitlg and Kit mRNA, pIRS1^Ser302, pAKT^Thr308, EPHX1, and GSTP1 protein level was observed due to obesity, while Cyp2e1 mRNA and protein were reduced. A phosphoramide mustard (PM) challenge increased (P < 0.05) ovarian EPHX1 protein abundance in lean but not obese females. In addition, lung tissue from PM-exposed animals had increased (P < 0.05) EPHX1 protein with no impact of obesity thereon. Taken together, progressive obesity affected ovarian signaling pathways potentially involved in obesity-associated reproductive disorders.

Summary Sentence

Obesity alters ovarian signaling pathways that regulate primordial follicle activation and chemical biotransformation, thereby potentially contributing to reproductive dysfunction.

Phosphoramide mustard (PM) destroys rapidly dividing cells and activates the DNA double strand break marker, γH2AX, and DNA repair in rat granulosa cells and neonatal ovaries. The effects of PM exposure on DNA damage and activation of DNA damage repair in lean and obese female mice were investigated. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice received sesame oil or PM (95%; 25 mg/kg; intraperitoneal injection). Obesity increased (P < 0.05) hepatic and spleen but decreased (P < 0.05) uterine weight. PM exposure reduced (P < 0.05) spleen weight regardless of body composition, however, decreased (P < 0.05) ovarian and hepatic weight were observed in the obese PM-exposed females. PM decreased (P < 0.05) primordial and primary follicle number in lean females. Obesity and PM increased (P < 0.05) γH2AX protein. DNA damage repair genes Prkdc, Parp1, and Rad51 mRNA were unaltered by obesity, however, Atm and Xrcc6 mRNA were increased (P < 0.05) while Brca1 was reduced (P < 0.05). Obesity reduced (P < 0.05) PRKDC, XRCC6 and but increased (P < 0.05) ATM protein. ATM, BRCA1 and RAD51 protein levels were increased (P < 0.05) by PM exposure in both lean and obese mice, while PMinduced increased (P < 0.05) XRCC6 and PARP1 were observed only in lean mice. Thus, PMinduces ovarian DNA damage in vivo; obesity alters DNA repair response gene mRNA and protein level; the ovary activates DNA repair proteins in response to PM; but obesity compromises the ovarian PM response.

Summary Sentence

PM exposure induces DNA damage and subsequent repair in ovaries of exposed mice, and this response is abrogated in obese females.