Coronavirus disease 2019 (COVID-19) is a multi-system disease that has led to a pandemic with unprecedented ramifications. The pandemic has challenged scientists for the past 2 years and brought back previously abandoned research topics. COVID-19 infection causes a myriad of symptoms ranging from mild flu-like symptoms to severe illness requiring hospitalization. Case reports showed multiple systemic effects of COVID-19 infection, including acute respiratory distress syndrome, fibrosis, colitis, thyroiditis, demyelinating syndromes, and mania, indicating that COVID-19 can affect most human body systems. Unsurprisingly, a major concern for women all over the globe is whether a COVID-19 infection has any long-term effects on their menstrual cycle, fertility, or pregnancy. Published data have suggested an effect on the reproductive health, and we hypothesize that the reported reproductive adverse effects are due to the robust immune reaction against COVID-19 and the associated cytokine storm. While the COVID-19 receptor (angiotensin converting enzyme, ACE2) is expressed in the ovaries, uterus, vagina, and placenta, we hypothesize that it plays a less important role in the adverse effects on the reproductive system. Cytokines and glucocorticoids act on the hypothalamo–pituitary gonadal axis, arachidonic acid pathways, and the uterus, which leads to menstrual disturbances and pregnancy-related adverse events such as preterm labor and miscarriages. This hypothesis is further supported by the apparent lack of long-term effects on the reproductive health in females, indicating that when the cytokine storm and its effects are dampened, the reproductive health of women is no longer affected.

Summary Sentence

COVID-19 affects the female reproductive system.

Cryopreserved ram spermatozoa are limited in their capacity to traverse the ovine cervix and achieve fertilization. This altered interaction may be related to modified molecular communication between frozen-thawed ram spermatozoa, seminal plasma, and the female tract. As such, this review aims to identify the biological processes which underpin sperm maturation and transport throughout the female reproductive tract to elucidate factors which may alter this natural process in cryopreserved ram spermatozoa. We also assess critical barriers to ram spermatozoa specific to the ovine cervix and the role of seminal plasma in mitigating these barriers. Transcriptomics is explored as a new approach to understand the sperm–cervix interaction. Recent studies have demonstrated that both spermatozoa and seminal plasma contain a complex profile of coding and non-coding RNAs. These molecular species have clear links with functional fertility, and mounting evidence suggests they may be altered by cryopreservation. Emerging in vitro cell culture models are also investigated as a “next step” in studying this interaction, utilizing transcriptomics to identify subtle changes in female tract gene expression in response to spermatozoa. The application of such models is proposed as an exciting opportunity to investigate the unique challenges faced by cryopreserved spermatozoa traversing the ovine cervix prior to fertilization.

Summary Sentence

Transcriptomics is suggested as a new approach to understand the sperm–cervix interaction which is thought to mediate the limited fertility associated with cervical artificial insemination and frozen-thawed ram spermatozoa.

Angiogenesis within the ovarian follicle is an important component of ovulation. New capillary growth is initiated by the ovulatory surge of luteinizing hormone (LH), and angiogenesis is well underway at the time of follicle rupture. LH-stimulated follicular production of vascular growth factors has been shown to promote new capillary formation in the ovulatory follicle. The possibility that LH acts directly on ovarian endothelial cells to promote ovulatory angiogenesis has not been addressed. For these studies, ovaries containing ovulatory follicles were obtained from cynomolgus macaques and used for histological examination of ovarian vascular endothelial cells, and monkey ovarian microvascular endothelial cells (mOMECs) were enriched from ovulatory follicles for in vitro studies. mOMECs expressed LHCGR mRNA and protein, and immunostaining confirmed LHCGR protein in endothelial cells of ovulatory follicles in vivo. Human chorionic gonadotropin (hCG), a ligand for LHCGR, increased mOMEC proliferation, migration and capillary-like sprout formation in vitro. Treatment of mOMECs with hCG increased cAMP, a common intracellular signal generated by LHCGR activation. The cAMP analog dibutyryl cAMP increased mOMEC proliferation in the absence of hCG. Both the protein kinase A (PKA) inhibitor H89 and the phospholipase C (PLC) inhibitor U73122 blocked hCG-stimulated mOMEC proliferation, suggesting that multiple G-proteins may mediate LHCGR action. Human ovarian microvascular endothelial cells (hOMECs) enriched from ovarian aspirates obtained from healthy oocyte donors also expressed LHCGR. hOMECs also migrated and proliferated in response to hCG. Overall, these findings indicate that the LH surge may directly activate ovarian endothelial cells to stimulate angiogenesis of the ovulatory follicle.

Summary Sentence The ovulatory surge of LH may regulate follicular angiogenesis by acting directly at LHCGRs on ovarian vascular endothelial cells.

Graphical Abstract

Mice are the most widely used animal model to study human diseases. However, the difficulty of in vivo recovery of mouse sperm has posed a limitation with its use in reproductive biology research. Several published techniques for obtaining sperm samples in vivo have been described, but most of them have several caveats. Critical limitations include poor reliability and significant mortality (Electroejaculation and drug-induced ejaculation), or the need for a large number of animals, careful programming, and laborious work (directed mating). Here, we describe a new approach for in vivo collection of sperm in the mouse via direct puncture of the epididymis to address these limitations. In addition, the technique is easy, safe, and reliable, allowing the animal to recover and maintain its fertility. In this way, punctual experiments could be carried out, or even more so, serial sampling of the same animal over time. Therefore, our approach allows for long-term and time-course experiments to study sperm characteristics under different treatments or conditions while maintaining the spermatogenic niche in vivo. In summary, we present our original approach as a powerful research tool to facilitate the study of spermatozoa relevant to various areas of biomedical research.

Summary Sentence The method for multiple sampling mouse sperm in vivo, allows for long-term and time-course experiments to study sperm characteristics under different treatments or conditions while maintaining the spermatogenic niche in vivo.

Olive flounder Paralichthys olivaceus is an important cultured marine fish. We found that the meiosis marker scp3 and its intrinsic regulator dazl were mainly expressed in the gonads. During the ovarian differentiation, scp3 signal was detected first in pre-meiotic oogonia at 60-mm total length (TL) and then in primary oocytes at 80- and 100-mm TL, with a sharp increase in scp3 expression level observed at 80- and 100-mm TL. Dazl signal was detected in primordial germ cells at 30-mm TL and oogonia at 60-mm TL, but no significant change of expression was observed. During the testicular differentiation period, scp3 and dazl expression remained at low levels, and scp3 signal was weakly detected in spermatogonia at 80-mm TL, whereas dazl signal was not found. During the ovarian developmental stages, the highest expression levels of scp3 and dazl were detected at stages I and II, respectively, and strong signals of scp3 and dazl were detected in primary oocytes and oocytes at phases I and II. In the testis, the high expression of scp3 and dazl was detected at stages II–IV and II–III, respectively. Scp3 signal was weakly observed in pre-meiotic spermatogonia at stages I and II and strongly detected in primary spermatocytes at stages III–V. Dazl was detected in the nuclei of spermatogonia and spermatids at stages II–IV. Furthermore, scp3 expression in the ovary could be promoted by 17α-ethynylestradiol and tamoxifen, whereas dazl expression could be downregulated by tamoxifen.

Summary Sentence

The research shows the meiosis begins in the flounder gonads later than the gonadal differentiation.

Graphical Abstract

Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of transcomplexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.

Summary Sentence: Cysteine string protein is necessary and mediates the trans-SNARE complexes assembly between the outer acrosomal and the plasma membranes in human sperm acrosomal exocytosis mechanism.

Graphical Abstract

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ∼D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoA^f/f) and P4-deficient RhoA^f/fPgr^Cre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoA^f/fPgr^Cre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoA^f/fPgr^Cre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.

Summary Sentence

Visualization of the temporal mechanisms of uterine fluid absorption via bulk entry and endocytosis during early pregnancy provides novel insights into cellular and molecular mechanisms in establishing uterine receptivity for embryo implantation.

Graphical Abstract

The tested hypotheses were (1) LH/FSH pulses and F2 diameter are diminished by P4 and, (2) E2 increases during the transition to deviation and alters LH/FSH pulses. On Day 5 (Day 0 = ovulation), heifers were randomized into an untreated group (HiP4, n = 11), and a prostaglandin analog treated group (NoP4, n = 10). On Day 6, a follicular wave was induced by follicle ablation. Ultrasound and blood collections were performed every 12 h from Days 7 to 11. Blood was collected every 15 min for 10 h on Day 9 (largest follicle expected to be ∼7.5 mm). Estradiol was ∼75% greater (0.36 ± 0.14 vs 0.63 ± 0.19 pg/mL) in heifers with F1 ≥ 7.2 mm than in heifers with F1 < 7.2 mm. The HiP4 had smaller second largest follicle (F2) diameter, lower estradiol (P = 0.06), LH pulse baseline and peak concentrations (P < 0.007), in addition to half the frequency of LH/FSH pulses (4.1 ± 0.3 vs 9.6 ± 0.7 in 10 h) than the NoP4. Within HiP4, heifers with F1 ≥ 7.2 mm had ∼25% fewer (P = 0.03) LH pulses compared to heifers with F1 < 7.2 mm. In contrast, within the NoP4, heifers with F1 ≥ 7.2 mm had ∼75% greater LH (P = 0.05) and FSH (P = 0.08) pulse amplitude. We propose that greater F2 diameter at deviation in low P4 is related to greater LH baseline and peak concentrations, and greater frequency of LH/FSH pulses. A greater increase in E2 after F1 reaches ∼7.2 mm results in further stimulation of LH/FSH pulse amplitude. Elevated P4 not only diminished frequency of LH/FSH pulses but also converted an E2 increase into a negative feedback effect on LH/FSH pulse frequency leading to smaller F2 at deviation.

Summary Sentence

Near follicle deviation, heifers in high P4 vs low P4 had smaller follicles and lower LH baseline, peak of the pulse, and frequency. Within high P4, E2 decreased LH/FSH pulse frequency. In contrast, within low P4, E2 increased LH/FSH pulse amplitude.

Spermatogenesis is a dynamic cell developmental process that is essential for reproductive success. Vertebrates utilize a variety of reproductive strategies, including sperm diversity, and internal and external fertilization. Research on the cellular and molecular dynamic changes involved in viviparous teleost spermatogenesis, however, is currently lacking. Here, we combined cytohistology, 10 × genomic single-cell RNA-seq, and transcriptome technology to determine the dynamic development characteristics of the spermatogenesis of Sebastes schlegelii. The expressions of lhcgr (Luteinizing hormone/Choriogonadotropin receptor), fshr (follicle-stimulating hormone receptor), ar (androgen receptor), pgr (progesterone receptor), and cox (cyclo-oxygen-ase), as well as the prostaglandin E and F levels peaked during the maturation period, indicating that they were important for sperm maturation and mating. Fifteen clusters were identified based on the 10 × genomic single-cell results. The cell markers of the sub-cluster were identified by their upregulation; piwil, dazl, and dmrt1 were upregulated and identified as spermatogonium markers, and sycp1/3 and spo11 were identified as spermatocyte markers. For S. schlegelii, the sperm head nucleus was elongated (spherical to streamlined in shape), which is a typical characteristic for sperm involved in internal fertilization. We also identified a series of crucial genes associated with spermiogenesis, such as spata6, spag16, kif20a, trip10, and klf10, while kif2c, kifap3, fez2, and spaca6 were found to be involved in nucleus elongation. The results of this study will enrich our cellular and molecular knowledge of spermatogenesis and spermiogenesis in fish that undergo internal fertilization.

Summary Sentence

In S. schlegelii, luteinizing hormone can induce prostanoid production owing to cox upregulation and prostaglandin may be a key pheromone in copulation and sperm maturation.

Successful attachment of conceptus to the uterine luminal epithelium (LE) is crucial for establishing a functional placenta in pigs. However, the underlying mechanisms are yet to be elucidated. The uterine LE-conceptus interface is enriched in various glycoconjugates essential to implantation. Using MALDI-MS profiling, we identified for the first time the O-glycan repertoire of pig endometrium during the conceptus attachment stage. The expression pattern of blood group A, O(H), Lewis x, y, a, b (Le^x, Le^y, Le^a, and Le^b), the sialylated and sulfated Le^x antigens in the uterine LE-conceptus interface was assessed using immunofluorescence assays. Notably, the Le^x-carrying O-glycans exhibited a temporal–spatial expression pattern. They were absent in the endometrium on estrous cycle days but strongly and spatially presented in the conceptus and uterine LE to which the conceptus apposes during the early conceptus attachment stage. In addition, Le^x-carrying O-glycans were co-localized with secreted phosphoprotein 1 (SPP1), a well-characterized factor that plays a role in promoting conceptus attachment through interacting with integrin αVβ3 and integrin αVβ6. Meanwhile, the immunoprecipitation assays revealed an interaction between the Le^x-carrying O-glycans and SPP1, integrin αV, and integrin β6. Furthermore, we provided evidence that the β1,4-galactosyltransferase 1 (B4GALT1) gene is a potential regulator for Le^x antigen expression in the uterine LE-conceptus interface during the early conceptus attachment stage. In conclusion, our findings show that Le^x-carrying O-glycans, presumably dependent on B4GALT1 gene expression, might modulate conceptus attachment by interacting with the SPP1-integrin receptor complex in pigs.

Graphical Abstract

Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production.

Summary Sentence

Transplantation of busulfan-resistant PGCs into the adult testes and enrichment of donor cells by eliminating endogenous germ cells with busulfan treatment of recipients improved the efficiency of germline chimera production in songbirds.

Environmental stressors to which a fetus is exposed affect a range of physiological functions in postnatal offspring. We aimed to determine the in utero effect of steroid hormones on the reproductive potential of female offspring using a porcine model. Reproductive tracts of pigs from female-biased (>65% female, n = 15), non-biased (45–54.9% female, n = 15), and male-biased litters (<35% females, n = 9) were collected at slaughter (95–115 kg). Ovaries and uterine horns were processed for H&E or immunohistochemistry. Variability of data within groups was analyzed with a Levene's test, while data were analyzed using mixed linear models in R. In the ovarian reserve, there was a significant birth weight by sex ratio interaction (P = 0.015), with low birth weight pigs from male-biased litters having higher numbers of primordial follicles with opposite trends seen in pigs from female-biased litters. Sex bias held no effect on endometrial gland development. A lower birth weight decreased the proportion of glands found in the endometrium (P = 0.045) and was more variable in both male-biased and female-biased litters (P = 0.026). The variability of primordial follicles from male-biased litters was greater than non- and female-biased litters (P = 0.014). Similarly, endometrial stromal nuclei had a greater range in male- and female-biased litters than non-biased litters (P = 0.028). A crucial finding was the greater variability in primordial follicles in the ovaries from females derived from male-biased litters and stromal cell count in the endometrium of females from male- and female-biased litters. This could be inflating the variability of reproductive success seen in females from male-biased litters.

Summary Sentence

Females gestated in utero in extremes of sex ratios have altered ovarian reserve postnatally but not uterine capacity.

Graphical Abstract

Mammalian folliculogenesis is a complex process that involves the regulation of chromatin structure for gene expression and oocyte meiotic resumption. The SWI/SNF complex is a chromatin remodeler using either Brahma-regulated gene 1 (BRG1) or BRM (encoded by Smarca4 and Smarca2, respectively) as its catalytic subunit. SMARCA4 loss of expression is associated with a rare type of ovarian cancer; however, its function during folliculogenesis remains poorly understood. In this study, we describe the phenotype of BRG1 mutant mice to better understand its role in female fertility. Although no tumor emerged from BRG1 mutant mice, conditional depletion of Brg1 in the granulosa cells (GCs) of Brg1^fl/fl; Amhr2-Cre mice caused sterility, whereas conditional depletion of Brg1 in the oocytes of Brg1^fl/fl;Gdf9-Cre mice resulted in subfertility. Recovery of cumulus-oocyte complexes after natural mating or superovulation showed no significant difference in the Brg1^fl/fl;Amhr2-Cre mutant mice and significantly fewer oocytes in the Brg1^fl/fl;Gdf9-Cre mutant mice compared with controls, which may account for the subfertility. Interestingly, the evaluation of oocyte developmental competence by in vitro culture of retrieved two-cell embryos indicated that oocytes originating from the Brg1^fl/fl;Amhr2-Cre mice did not reach the blastocyst stage and had higher rates of mitotic defects, including micronuclei. Together, these results indicate that BRG1 plays an important role in female fertility by regulating granulosa and oocyte functions during follicle growth and is needed for the acquisition of oocyte developmental competence.

Summary Sentence

Conditional depletion of Brahma-regulated gene 1 in granulosa cells early in follicle development and in oocytes results in sterile and subfertile phenotypes, respectively.

Graphical Abstract

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2^Cre; IFT88^fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Summary Sentence

Modulation of primary-ciliogenesis and downstream Hedgehog signaling controls Wolffian duct development.

Although recent research has addressed the impact of cryopreservation on the stallion sperm proteome, studies addressing the stallion sperm phosphoproteome are lacking. In the present study, the data set of proteomes of fresh and cryopreserved spermatozoa were reanalyzed, showing that cryopreservation caused significant changes in the phosphoproteome. The phosphoproteins reduced most significantly by cryopreservation were Ca²⁺ binding tyrosine phosphorylation regulated, protein kinase cAMP-activated catalytic subunit beta (CABYR), mitochondria eating protein (SPATA18), A kinase anchoring protein 4 (AKAP4), A-kinase anchoring protein 3 (AKAP3) and the Family with sequence similarity 71 member B (FAM71B). These proteins belong to the gene ontology (GO) terms sperm fibrous sheath (GO: 0035686), and sperm principal piece (GO: 0097228). The regulatory interactions between kinases and phosphorylation sites on the proteins that were affected most were also investigated, and the potential kinases (based on human orthologs) involved in the regulation of these phosphoproteins identified were: PKCß for SPATA18 and GSK3ß for CABYR. Kinase inhibition assays were also conducted showing that kinases phosphorylating the above-mentioned proteins play an important role in their activity and thus, phosphorylation controls the activity of these proteins and their role in the regulation of the functionality and viability of stallion spermatozoa. In conclusion, the data reported here contribute to the understanding of the fact that the dephosphorylation of certain proteins is a molecular lesion induced by cryopreservation in the stallion spermatozoa.

Graphical Abstract